Lipid nanoparticles for delivery of sting-dependent adjuvants

ABSTRACT

Activation of STimulator of INterferon Genes (STING) triggers cytokine production and facilitates tumor antigen cross-presentation. In an embodiment of the present invention, STING-dependent innate immune signaling pathway activators (STAVs) can be delivered to antigen presenting cells. In various embodiments of the present invention, the STAVs can be delivered in lipid nanoparticle formulations. In various embodiments of the present invention, the range of cancers amenable to STAV therapy can be extended using a non-cell-based nanoparticle strategy that effectively delivers Nano-STAVs into the Tumor Micro Environment (TME) to potently generate anti-tumor cytotoxic T cell activity. The STAV formulations can be introduced into solid tumors present in the mammal. Alternatively, the Nano-STAVs can be introduced through direct inoculation. The lipid nanoparticles stick to the tumor cells and are co-phagocytosed to activate STING in APC&#39;s.

PRIORITY CLAIM

This application claims priority to and is a continuation in part of (1)U.S. application Ser. No. 16/621,820 filed Dec. 12, 2019, entitledSTING-DEPENDENT ACTIVATORS FOR TREATMENT OF DISEASE, by Glen N. Barberwhich is the national phase of (2) PCT Application No. PCT/US2018/036997entitled “STING-Dependent Activators for Treatment of Disease”, inventorGlen N. Barber, filed Jun. 12, 2018, which claims priority to (3) U.S.Provisional Patent Application No. 62/518,292 filed Jun. 12, 2017 andthis application also claims priority to and is a continuation in partof (4) PCT Application No. PCT/US22/034796 entitled “Lipid nanoparticlevector for delivery of Sting Dependent Adjuvants (STAVS)”, inventor GlenN. Barber, filed Jun. 23, 2022, which claims priority to (5) U.S.Provisional Patent Application No. 63/214,671, filed Jun. 24, 2021 and(6) U.S. Provisional Patent Application No. 63/349,004, filed Jun. 24,2022, and (7) U.S. Provisional Patent Application No. 63/354,199, filedJun. 21, 2022 which applications (1)-(7) are herein incorporated byreference in their entireties and for all purposes.

The Sequence Listing in WIPO Standard ST.26 form written in fileSTNG-01014US0_ST26.xml, created Feb. 28, 2023, 99, 119 bytes, machineformat IBM-PC, MS-Windows operating system, is hereby incorporated byreference in its entirety and for all purposes.

FIELD OF THE INVENTION

Embodiments of the invention relate to compositions and methods formodulating innate and adaptive immunity in a subject and/or for thetreatment of an immune-related disorder, cancer, autoimmunity, treatingand preventing infections with Sting Dependent Adjuvants.

BACKGROUND OF THE INVENTION

Cellular host defense responses to pathogen invasion principallyinvolves the detection of pathogen associated molecular patterns (PAMPs)such as viral nucleic acid or bacterial cell wall components includinglipopolysaccharide or flagellar proteins that results in the inductionof anti-pathogen genes. For example, viral Ribonucleic Acid (RNA) can bedetected by membrane bound Toll-like receptors (TLR's) present in theEndoplasmic Reticulum (ER) and/or endosomes (e.g. Toll-like receptor 3(TLR 3) and TLR7/TLR8) or by TLR-independent intracellular DExD/H boxRNA helicases referred to as Retinoic acid Inducible Gene 1 (RIG-1) orMelanoma Differentiation associated Antigen 5 (MDA5), also referred toas IFIH1 and helicard. These events culminate in the activation ofdownstream signaling events, much of which remains unknown, leading tothe transcription of Nuclear Factor kappa-light-chain-enhancer ofactivated B cells (NF-κB) and Interferon Regulatory Factor 3(IRF3)/IRF7-dependent genes, including type I Interferon (IFN).

ATLL was first described as a distinct clinical entity in 1979 and itsassociation with the human T-cell leukemia virus type 1 (HTLV-1) wasreported shortly thereafter. HTLV-1 affects about 10-20 million peopleworldwide and is endemic in southwest Japan, sub-Saharan Africa, theCaribbean, and parts of South America, particularly Brazil and Peru.South Florida (containing Miami and Broward counties) due to its closeproximity to the Caribbean, has a large population of immigrants fromHTLV-1 endemic areas, therefore ATLL is commonly encountered in thisgeographic area. African-American patients are also frequently diagnosedwith ATLL at the University of Miami and Jackson Memorial Hospital. ATLLpatients are also frequently encountered in New York City. ATLL canpresent in multiple forms and is generally sub-classified into foursubtypes. Lymphoma and acute ATLL are the two most aggressive variantswhere patients usually present with a high tumor burden andhypercalcemia. The chronic and smoldering forms of ATLL have a moreindolent course, although they often progress to the more malignantforms of the disease. ATLL carries a dismal prognosis, and is generallyincurable with conventional chemotherapy alone. In a Japanese study of1,594 patients with ATLL treated with modern aggressive therapiesbetween 2000 and 2009, the median survival (MS) times were 8.3 and 10.6months for acute and lymphomatous types respectively. A subset ofpatients with leukemic types of ATLL with non-bulky tumors or lymphnodes may benefit long-term from AZT-interferon-α therapy, however, thistreatment is only suppressive and all patients ultimately relapse andsuccumb to their disease. A relatively small group of patients becomeeligible for allogeneic stem cell transplant (allo-HSCT) with thepossibility of long-term cure. Despite this, in the Katsuya study, 227patients who underwent allo-HSCT were included, and the MS was only 5.9months with 4-yr survival of 26%.

AML is the most common form of acute leukemia in adults and accounts forthe largest number of deaths from leukemias in the United States. Over20,000 people are diagnosed with AML per year, and roughly half of thisnumber die of it each year. AML usually affects older patients with themedian age at diagnosis at 67 years. Standard induction chemotherapyregimens are used for patients younger than age 60 consisting of abackbone of cytarabine plus an anthracycline. Complete response ratesfor patients who are 50 years or younger are in the range of 60% to 70%but most patients ultimately relapse and succumb to their disease. Poorperformance status and medical co-morbidities limit the ability toadminister aggressive standard therapy to older patients and those thatdo get treated often receive suboptimal treatment. In elderly patientstreated with intensive chemotherapy there has been little improvement insurvival indicating the need for alternative approaches. Currently, the5-year overall survival for AML hovers around 25%, while in patients 65or older it is <10%.

B-cell or T-cell ALL is the least common type of acute leukemia inadults although it is the most common in the pediatric population. Adultpatients have a relatively poor prognosis as compared to children andyoung adults, who can be cured with intensive chemotherapy. Prognosisvaries according to disease presentation and molecular subtypes. Forinstance, the 5-year overall survival rate among adult patients withPhiladelphia chromosome-positive (Ph+) pre-B-cell ALL is only 25%. Therelapsed disease setting is often fatal in adults.

STimulator of Interferon Genes (STING) is a 379 amino acid transmembraneprotein located in the cytosol endoplasmic reticulum of for examplefibroblasts, macrophages and DCs. STING is a DNA sensor that has evolvedto detect microbial infection of the cell. STING is activated by cyclicdinucleotides (CDN's) such as cyclic di-GMP and cyclic-di-AMP secretedby intracellular bacteria following infection. Alternatively, STING canbe activated by cyclic GMP-AMP (cGAMP) generated by a cellular cGAMPsynthase cGAS (MB21D1) after association with aberrant cytosolic dsDNAspecies, which can include microbial DNA or self-DNA leaked from thenucleus into the cytosol. CDN-binding results in STING, complexed withthe IRF3 kinase TANK-binding kinase 1 (TBK1) re-locating to perinuclearregions of the cell. Association with CDN's enables STING to activatethe transcription factors IRF3 and NF-κB which stimulate the productionof type I interferon (IFN) and pro-inflammatory cytokines, whichfacilitate adaptive immunity.

Lipid nanoparticle technology offers the promise of high nucleic acidencapsulation efficiency, potent transfection and improved penetrationof Sting Dependent Adjuvants (STAVs) into cells with low cytotoxicityand immunogenicity.

Programmed death-ligand 1 (PD-L1) together with its receptor programmedcell death protein 1 (PD-1) are ‘check point’ proteins involved in theregulation of the immune response. The interaction of these cell surfaceproteins can for example suppress the immune system following infectionto limit the killing of bystander host cells. These checkpoint proteinscan be used by some types of cancer to block the immune system's abilityto attack the cancerous cells.

PD-L1 inhibitors and PD-1 inhibitors are check point inhibitors of PD-L1and PD-1 respectively and act to inhibit the association of the PD-L1with PD-1. By blocking the activity of PD-L1 and PD-1 the inhibitors canbe used to restore the immune system's ability to attack the cancerouscells and therefore used as anticancer drugs.

One in five Americans will develop skin cancer by the age of seventy. Itis the most common of all solid tumors. One of the most serious skincancers is melanoma, where an estimated 200,000 people will be diagnosedin the USA in 2021. A variety of treatments are available, includingvaccines, kinase inhibitors as well as oncolytic viruses (T-VEC;Imlygic, talimogene laherparevec) with varying response rates. In 2015,the FDA approved T-VEC for patients with advanced melanoma (Stage IIIB,IIIC or IV) that could not be removed with surgery and were refractoryto alternate treatments. T-VEC is injected cutaneous, sub-cutaneous ordirectly into nodes.

SUMMARY OF THE INVENTION

Tumor cells are notoriously non-immunogenic through their ability tomimic the properties of normal cells which have naturally evolved toavoid activating the immune system following cell death andphagocytosis. In an embodiment of the present invention, a new approachovercomes this obstacle and makes previously immuno-evasive, inert tumorcells highly immunogenic. This has been achieved by developingDNAse-resistant nucleic acid-based STING-dependent adjuvants oractivators, referred to as STAVs (dsDNA species of approximate length 76nucleotides) as activators of the STING-dependent innate immunesignaling pathway. In an embodiment of the present invention, syngeneictumor cells loaded with STAVs rendered non-immunogenic cellsimmunogenic. In an embodiment of the present invention, the syngeneictumor cells loaded with STAVs are able to stimulate antigen presentingcells (APCs) in vitro and in vivo. Immunocompetent mice bearingmetastatic, melanoma tumors could be cured following inoculation ofsyngeneic tumor cells loaded with STAVs. In an embodiment of the presentinvention, syngeneic tumor cells loaded with STAVs ex vivo can be usedto treat autologous aggressive leukemia cells (ATLL, AML, and ALL)concomitant with a personalized dendritic cell (DCs) vaccine (preparedfrom DCs stimulated by dead UV-irradiated STAVs loaded leukemic cells).

DCs are specialized APCs found in blood and throughout most organtissues. DCs strongly express major histocompatibility complex (MHC),adhesion, and co-stimulatory molecules necessary for the stimulation ofT cell responses and adaptive cell immunity. DCs are located at sites ofantigen capture and after they phagocyte pathogens, foreign antigens, ordamaged cells they subsequently migrate to lymphatic areas for antigenpresentation. By expressing both MHC class I and class II molecules,they can prime both cytotoxic CD8+ cells and CD4+ helper T-cellsrespectively, and both of these cell types are thought to be necessaryfor an effective cell-mediated immune response. DCs can also stronglyactivate NK and NK-T cells thus linking innate and adaptive immuneresponses thus potentially targeting tumor cells for killing with andwithout expression of MHC class I molecules. DCs have been demonstratedto interact with foreign antigens ex vivo, present these to naïve CD4C Tcells, and to generate clonal expansion of effector T cells.

DC vaccines have emerged as promising cancer immunotherapy approach. DCvaccines can be generated from large numbers of progenitor cellscultured ex vivo in the presence of cytokines after exposing these toforeign antigens. Tumor cells can evade immune recognition by blunting Tcell responses via several mechanisms; these may include: 1) presentingtumor antigens in the relative absence of co-stimulatory moleculesrequired for the activation of effector T cells thus inducing T cellanergy rather than immunity, 2) creating a micro-environment rich inimmunosuppressive T-regulatory cells (Tregs) and myeloid derivedsuppressor cells, and 3) upregulating negative co-stimulatory pathwayssuch as those mediated by CTLA-4 and PDL-1/PD-1 thus favoring tumorgrowth and survival. Malignant cells can also inhibit the function ofDCs thus making them more tolerant to tumor antigens. In an embodimentof the present invention, an effective cancer vaccine requires efficientpresentation of tumor antigens, adequate co-stimulation leading toT-cell priming, and successful reversal of the immunosuppression inducedby tumor cells in order to achieve long-term immunity. Animal modelshave demonstrated that DC tumor vaccines can reverse T-cell anergyresulting in subsequent tumor rejection.

Clinical trials and pre-clinical studies have evaluated DC vaccinesagainst various cancers, including hematologic malignancies, anddemonstrated safety. In one study, a personalized whole tumor cell(AML)/DC fusion vaccine elicited the expansion of leukemia-specific Tcells and protected against disease relapse in elderly patients withAML. A recently developed HTLV-1 Tax-DC vaccine consisting of autologousDCs pulsed with Tax peptides corresponding to CTL epitopes wasadministered to three pre-treated ATLL patients, and two patientssurvived for more than 4 years after vaccination without severe adverseeffects. DCs loaded with leukemia-derived apoptotic bodies from adultpatients with ALL increased their ability to stimulate both allogeneicand autologous T lymphocytes, and to generate specific anti-leukemicCD3+ cells. These findings offered a rationale for designing DC-basedvaccines for patients with ALL with the objective ofcontrolling/eradicating the disease. In an embodiment of the presentinvention, the paradigm will include personalized serial injections ofautologous mature DCs stimulated exogenously with patient's own STAVsloaded leukemic cells.

ATLL is a clonal disease triggered by HTLV-1 infection that isinvariably lethal and for which there is no cure or vaccine.Relapsed/refractory AML and ALL in adult patients are also incurable andoften rapidly fatal despite aggressive treatment. Recent clinical trialstesting the use of adjuvant vaccination with antigen stimulatedautologous mature DCs have shown that DC vaccination is safe, feasible,and potentially beneficial for patients. The stimulation of innateimmune signaling pathways leading to cytokine production withinphagocytes such as CD8+ DCs involve STING. In an embodiment of thepresent invention, a new generation of innate immune activators thattrigger STING signaling are referred to as STAVs: STING dependentadjuvants or activators. Tumor cells transfected with STAVs activateAPCs in trans and generate potent anti-tumor T cell activity. Theability of dying cells to activate APCs is carefully controlled to avoidunwarranted inflammatory responses. Thus, dying tumor cells avoidaggravating APCs which following phagocytosis do not triggerinflammatory responses required for efficient CTL priming. However,dying tumor cells containing exogenous innate immune agonists such ascytosolic DNA, escape anti-inflammatory defenses and potently activateAPCs in trans through extrinsic innate immune, STING-dependent signalingto generate potent CTL activity. In the absence of STING agonists, dyingcells are ineffectual in the stimulation of APCs in trans. Indeed,cytosolic STING activators, including cytosolic DNA and cyclicdinucleotides (CDNs), constitute cellular danger associated molecularpatterns (DAMPs) (usually only generated by viral infection or followingDNA-damage events) that can render tumor cells highly immunogenic.

STING, a molecule that plays a key role in the innate immune response,includes 5 putative transmembrane (TM) regions, predominantly resides inthe endoplasmic reticulum (ER), and is able to activate both NF-κs andIRF3 transcription pathways to induce type I IFN and to exert a potentanti-viral state following expression (see U.S. patent application Ser.No. 16/717,325 and PCT/US2009/052767 each of which is incorporatedherein by reference in its entirety and for all purposes). Loss of STINGreduced the ability of Polyinosinic:polycytidylic acid (polyIC) toactivate type I IFN and rendered Murine Embryonic Fibroblasts (MEFs)lacking STING (^(−/−) MEFs) generated by targeted homologousrecombination, susceptible to vesicular stomatitis virus (VSV)infection. In the absence of STING, DNA-mediated type I IFN responseswere inhibited, indicating that STING may play an important role inrecognizing DNA from viruses, bacteria, and other pathogens which caninfect cells. Yeast-two hybrid and co-immunoprecipitation studiesindicated that STING interacts with RIG-1 and with Ssr2/TRAPβ, a memberof the translocon-associated protein (TRAP) complex required for proteintranslocation across the ER membrane following translation. RNAiablation of TRAPP inhibited STING function and impeded the production oftype I IFN in response to polyIC.

Further experiments showed that STING itself binds nucleic acidsincluding single- and double-stranded DNA such as from pathogens andapoptotic DNA, and plays a central role in regulating pro-inflammatorygene expression in inflammatory conditions such as DNA-mediatedarthritis and cancer. Various new methods of, and compositions for,upregulating STING expression or function are described herein alongwith further characterization of other cellular molecule which interactwith STING. These discoveries allow for the design of new adjuvants,vaccines and therapies to regulate the immune system and other systems.

In one aspect, the present application relates to a composition fortreating a human subject suffering from cancer comprising a Nano-STAVcomprising a double-stranded DNA; and a lipid nanoparticle comprising apolymer-conjugated lipid, a sterol, a phospholipid; and an ionizinglipid.

In one aspect, the present application relates to a composition fortreating a human subject suffering from cancer comprising a firstNano-STAV comprising a first STAV selected from the group consisting ofSTAV1=(SEQ ID NO:24)+(SEQ ID NO:25); STAV2=(SEQ ID NO:26)+(SEQ IDNO:27); and STAV3=(SEQ ID NO:37)+(SEQ ID NO:38), and a LNP comprising apolymer-conjugated lipid, a sterol, a phospholipid, and an ionizinglipid.

In one aspect, the present application relates to a composition fortreating a human subject suffering from cancer comprising a firstNano-STAV comprising a first STAV selected from the group consisting ofSTAV1=(SEQ ID NO:24)+(SEQ ID NO:25); STAV2=(SEQ ID NO:26)+(SEQ IDNO:27); and STAV3=(SEQ ID NO:37)+(SEQ ID NO:38), and a LNP comprising apolymer-conjugated lipid selected from the group consisting of DMG-PEG2000 and DSPE PEG 2000, cholesterol, distearoylphosphatidylcholine, and(6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate.

In another aspect, the present application relates to a pharmaceuticalcomposition comprising a therapeutically effective amount of thecomposition of the application and a pharmaceutically acceptablecarrier.

In an embodiment of the present invention, a method of modulating (e.g.,inhibiting or stimulating) a STING protein involves application of thecomposition of the application or the pharmaceutical composition. Themethod comprises administering to a subject in need thereof an effectiveamount of the composition of the application or a pharmaceuticallyacceptable salt or ester thereof, or a pharmaceutical composition of theapplication. In one embodiment, the STING protein is a human STINGprotein.

In an embodiment of the present invention, a method of treating orpreventing a disease, wherein the disease is caused by, or associatedwith, STING expression, activity, and/or function (e.g., deregulation ofSTING expression, activity, and/or function) involves application of thecomposition of the application. The method further comprisesadministering to a subject in need thereof an effective amount of thecomposition of the application or a pharmaceutically acceptable salt orester thereof, or a pharmaceutical composition of the application.

In an embodiment of the present invention, a method of treating orpreventing a disease associated with deregulation of one or more of theintracellular pathways in which a STING protein is involved (e.g.,deregulation of intracellular dsDNA mediated type I interferonactivation). The method comprises administering to a subject in needthereof an effective amount of the composition of the application or apharmaceutically acceptable salt or ester thereof, or a pharmaceuticalcomposition of the application.

In an embodiment of the present invention, a kit comprising thecomposition of the application or a pharmaceutically acceptable salt orester thereof, or a pharmaceutical composition of the application.

In an embodiment of the present invention, a compound of the applicationor a pharmaceutically acceptable salt or ester thereof, or apharmaceutical composition of the application, for use in themanufacture of a medicament for modulating (e.g., inhibiting orstimulating) a STING protein, for treating or preventing a disease,wherein the diseases is caused by, or associated with, STING expression,activity, and/or function (e.g., deregulation of STING expression,activity, and/or function), or for treating or preventing a diseaseassociated with deregulation of one or more of the intracellularpathways in which a STING protein is involved (e.g., deregulation ofintracellular dsDNA mediated type I interferon activation).

In an embodiment of the present invention, use of the composition of theapplication or a pharmaceutically acceptable salt or ester thereof, or apharmaceutical composition of the application, in the manufacture of amedicament for modulating (e.g., inhibiting or stimulating) a STINGprotein, for treating or preventing a disease, wherein the diseases iscaused by, or associated with, STING expression, activity, and/orfunction (e.g., deregulation of STING expression, activity, and/orfunction), or for treating or preventing a disease associated withderegulation of one or more of the intracellular pathways in which aSTING protein is involved (e.g., deregulation of intracellular dsDNAmediated type I interferon activation).

In an embodiment of the present invention, the composition of theapplication or a pharmaceutically acceptable salt or ester thereof, or apharmaceutical composition of the application, for use in modulating(e.g., inhibiting or stimulating) a STING protein, in treating orpreventing a disease, wherein the diseases is caused by, or associatedwith, STING expression, activity, and/or function (e.g., deregulation ofSTING expression, activity, and/or function), or in treating orpreventing a disease associated with deregulation of one or more of theintracellular pathways in which a STING protein is involved (e.g.,deregulation of intracellular dsDNA mediated type I interferonactivation).

In an embodiment of the present invention, use of the composition of theapplication or a pharmaceutically acceptable salt or ester thereof, or apharmaceutical composition of the application, in modulating (e.g.,inhibiting or stimulating) a STING protein, in treating or preventing adisease, wherein the diseases is caused by, or associated with, STINGexpression, activity, and/or function (e.g., deregulation of STINGexpression, activity, and/or function), or in treating or preventing adisease associated with deregulation of one or more of the intracellularpathways in which a STING protein is involved (e.g., deregulation ofintracellular dsDNA mediated type-1 interferon activation).

The present application provides nano-STAVs that are therapeutic agentsin the treatment or prevention of diseases such as cancer inflammation,and other immunological disorders.

The details of the disclosure are set forth in the accompanyingdescription below. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent application, illustrative methods and materials are nowdescribed. In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and are not intended to be limiting.Other features, objects, and advantages of the disclosure will beapparent from the description and from the claims. In the specificationand the appended claims, the singular forms also include the pluralunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the present invention will be described in detailbased on the following Figures, where:

FIG. 1 is a line drawing representation showing the confocal analysis ofB16 OVA cells transfected with no DNA, labeled with DAPI 210 andanti-calreticulin 205;

FIG. 2 is a line drawing representation of showing confocal analysis ofB16 OVA cells transfected with STAVs-FAM, labeled with FAM 215 DAPI 210and anti-calreticulin 205;

FIG. 3A shows flow cytometry analysis of B16 OVA cells transfected withno DNA;

FIG. 3B shows flow cytometry analysis of B16 OVA cells transfected withSTAVs-FAM;

FIG. 4A shows a Transmission Electron Microscopy image of Nano-EmptyLNPs at high magnification;

FIG. 4B shows a Transmission Electron Microscopy image of Nano-STAV LNPsat high magnification;

FIG. 4C shows a Transmission Electron Microscopy image of Nano-EmptyLNPs at low magnification;

FIG. 4D shows a Transmission Electron Microscopy image of Nano-STAV LNPsat low magnification;

FIG. 4E shows a plot of cytokine expression for CXCL10 measured withqPCR in WT bone marrow derived mouse macrophages (11=PBS, 32=STAV,33=STAV+lipofectamine, 34=Nano-Empty, and 35=Nano-STAV);

FIG. 4F shows a plot of cytokine expression for CXCL10 measured withqPCR in SKO bone marrow derived mouse macrophages (11=PBS, 32=STAV,33=STAV+lipofectamine, 34=Nano-Empty, and 35=Nano-STAV);

FIG. 4G shows a plot of cytokine expression for CXCL10 measured withqPCR in MAVS KO bone marrow derived mouse macrophages (11=PBS, 32=STAV,33=STAV+lipofectamine, 34=Nano-Empty, and 35=Nano-STAV), according tovarious embodiments of the present invention;

FIG. 4H shows a plot of cytokine expression for IFN3 measured with ELISAin WT bone marrow derived mouse macrophages (11=PBS, 32=STAV,33=STAV+lipofectamine, 34=Nano-Empty, and 35=Nano-STAV), according tovarious embodiments of the present invention;

FIG. 4I shows a plot of cytokine expression for IFN3 measured with ELISAin SKO bone marrow derived mouse macrophages (11=PBS, 32=STAV,33=STAV+lipofectamine, 34=Nano-Empty, and 35=Nano-STAV), according tovarious embodiments of the present invention;

FIG. 4J shows a plot of cytokine expression for IFN3 measured with ELISAin MAVS KO bone marrow derived mouse macrophages (11=PBS, 32=STAV,33=STAV+lipofectamine, 34=Nano-Empty, and 35=Nano-STAV), according tovarious embodiments of the present invention;

FIG. 4K shows a plot of cytokine expression for CXCL10 measured withqPCR in DCs (11=PBS, 32=STAV, 33=STAV+lipofectamine, 34=Nano-Empty, and35=Nano-STAV), according to various embodiments of the presentinvention;

FIG. 4L shows a plot of IFN3 measured with ELISA in DCs (11=PBS,32=STAV, 33=STAV+lipofectamine, 34=Nano-Empty, and 35=Nano-STAV),according to various embodiments of the present invention;

FIG. 5A shows tumor volume of mice s.c. injected with B16 OVA cells(5×10⁵ cells/mouse) on the right flank. On day 7, 10, and 13 after tumorinoculation, the mice were i.t. injected with 11=PBS, 32=STAV,34=Nano-Empty, and 35=Nano-STAV (0.1 μg/mouse). The tumor volume wasmeasured and calculated with the formula V=(length×width²)/2. At 17days, the spleen was extracted to measure IFNγ release from CD8+ Tcells, according to various embodiments of the present invention;

FIG. 5B shows digital photographs of mice treated as in FIG. 5A,according to various embodiments of the present invention;

FIG. 5C shows IFNγ-ELISPOT of (OVA−) mice treated as in FIG. 5A;

FIG. 5D shows IFNγ-ELISPOT of (OVA+) mice (i.e., s.c. injected with B16OVA cells (5×10⁵ cells/mouse) on the right flank) and treated as in FIG.5A, according to various embodiments of the present invention;

FIG. 5E shows tumor volume of mice treated as in FIG. 5A or 36=HSV1-γ34.5;

FIG. 6A shows tumor volume of mice treated as in FIG. 4A with 11=PBS,32=STAV, 37=anti-PD1 (50 μg/mice) administered i.p., 34=Nano-Empty,38=Nano-Empty+anti-PD1 (50 μg/mice) administered i.p., 35=Nano-STAV (0.1μg/mouse), and 39=Nano-STAV+anti-PD1 (50 μg/mice) administered i.p.,according to various embodiments of the present invention;

FIG. 6B shows digital photographs of mice treated as in FIG. 6A,according to various embodiments of the present invention;

FIG. 6C shows IFNγ-ELISPOT of (OVA−) mice treated as in FIG. 6A;

FIG. 6D shows IFNγ-ELISPOT of (OVA+) mice (i.e., s.c. injected with B16OVA cells (5×10⁵ cells/mouse) on the right flank) and treated as in FIG.6A, according to an embodiment of the present invention;

FIG. 7A a flow diagram showing the protocol for administration ofNano-STAVs according to an embodiment of the present invention;

FIG. 7B a flow diagram showing the protocol for administration ofNano-STAVs according to an embodiment of the present invention;

FIG. 7C a flow diagram showing the protocol for administration ofNano-STAVs with check point inhibitors according to an embodiment of thepresent invention;

FIG. 7D a flow diagram showing the protocol for administration ofNano-STAVs with check point inhibitors according to an embodiment of thepresent invention;

FIG. 8A is a histogram showing an IFN3 ELISA assay in mouse embryonicfibroblasts (MEFs) Wild Type (WT) or STING Knock Out (SKO) cellstransfected with different lengths of AT rich-STING ligands(lipofectamine 2000 transfection reagent only 11; A:T30ES 22 (SEQ IDNO:1, SEQ ID NO:2); A:T50ES 23 (SEQ ID NO:3, SEQ ID NO:4); A:T60ES 24(SEQ ID NO:5, SEQ ID NO:6); A:T70ES 25 (SEQ ID NO:7, SEQ ID NO:8);A:T80ES 26 (SEQ ID NO:9, SEQ ID NO:10); A:T90ES (27 (SEQ ID NO:11, SEQID NO:12); and A:T100ES 28 (SEQ ID NO:13, SEQ ID NO:14));

FIG. 8B is a histogram showing an IFN3 ELISA assay in hTERT fibroblaststransfected with different lengths of AT rich-STING ligands (11; A:T30ES22, A:T50ES 23, A:T60ES 24; A:T70ES 25; A:T80ES 26; A:T90ES 27; andA:T100ES 28);

FIG. 8C is a histogram showing a quantitative Real Time-Polymerase ChainReaction (qRT-PCR) analysis of IFN31 in human macrophages transfectedwith different length of AT rich-STING ligands (11; A:T30ES 22; A:T50ES23; A:T60ES 24; A:T70ES 25; A:T80ES 26; A:T90ES 27; A:T100ES 28); andA:T110ES 29 (SEQ ID NO:15, SEQ ID NO:16));

FIG. 8D is a histogram showing an IFN3 ELISA assay in MEFs WT or SKOcells transfected with different lengths of GC rich-STING ligands (11;GC30ES 32 (SEQ ID NO:17); GC50ES 33 (SEQ ID No:18); GC60ES 34 (SEQ IDNO:19); GC70ES 35 (SEQ ID NO:20); GC80ES 36 (SEQ ID NO:21); GC90ES 37(SEQ ID NO:22); and GC100ES 38 (SEQ ID NO:23));

FIG. 8E is a histogram showing an IFNβ ELISA assay hTERT fibroblasttransfected with different lengths of GC rich-STING ligands (11; GC30ES32; GC50ES 33; GC60ES 34; GC70ES 35; GC80ES 36; GC90ES 37; and GC100ES38);

FIG. 8F is a histogram showing a qRT-PCR analysis of IFNβ1 in humanmacrophages transfected with different length of GC rich-STING ligands(11; GC30ES 32; GC50ES 33; GC60ES 34; GC70ES 35; GC80ES 36; GC90ES 37;and GC100ES 38);

FIG. 9A shows the growth in tumor volumes from WT (n=7/group) mice s.c.injected with murine B16 melanoma cells (B16-OVA cells) on the flank andsubsequently injected i.t. every 3 days with 10 μg of STAVs (STAV1=SEQID NO:24, SEQ ID NO:25; STAV2=SEQ ID NO:26, SEQ ID NO:27; STAV3=SEQ IDNO:28, SEQ ID NO:29; STAV4=, SEQ ID NO:30, SEQ ID NO:31; STAV5=(SEQ IDNO:32, SEQ ID NO:33) or Phosphate Buffered Saline (PBS) (as a control)and measured on the indicated days;

FIG. 9B shows the growth in tumor volumes from SKO (n=7/group) mice s.c.injected with murine B16 melanoma cells (B16-OVA cells) on the flank andsubsequently injected i.t. every 3 days with 10 μg of STAVs or PBS (ascontrol) and measured on the indicated days;

FIG. 9C is a histogram showing the frequency of OVA specific CD8+ Tcells (using OVA257-264 (SIINFEKL) peptide, SEQ ID NO:34) in the spleenfrom: WT (n=4/group) mice injected with PBS as control 12, withSTAV1-STAV5 44; SKO (n=4/group) mice injected with PBS as control 13,with STAV1 45;

FIG. 10A shows tumor volumes of mice treated as follows: on Day 0, C1498cells were s.c. inoculated in wild type C57/BL6 mice, where the C1498cells (AML tumor cells) were transfected with STAVs (3 μg/ml) for 3hours and irradiated by UV (120 mJ/cm for 1 minute) and incubated for 24hours. The mice were intraperitoneally (i.p.) injected with theirradiated C1498 cells with/without STAVs three times, STAV1 on Day 2,STAV2 on Day 5, and STAV3 on Day 10; and measured on the indicated days,where the tumor size from mice treated with C1498 cells with PBS 12,mice treated with C1498 cells without STAVs 45, and mice treated withC1498 containing STAVs 44 were measured;

FIG. 10B shows the tumor weight measured on Day 16 of the mice treatedas per FIG. 3A;

FIG. 10C shows an indirect ELISA analysis of plates pre-coated withSTAV1 at 0.1 μg/ml, where serum from mice treated with C1498 cells withPBS 12, mice treated with C1498 cells without STAVs 45, and mice treatedwith C1498 containing STAVs 44 was added to the ELISA plate wells.Anti-dsDNA (Abcam: ab27156) 14 was used as calibrator (standard curve),mice treated with control cells (not C1498 cells AML tumor) were used asa control 13.

FIG. 10D shows flow cytometry analysis of splenocytes isolated on Day 16and stained with anti-CD19-Alexa Fluor 700, where splenocytes from micetreated with C1498 cells with PBS 12, mice treated with C1498 cellswithout STAVs 45, and mice treated with C1498 containing STAVs 44, micetreated with anti-dsDNA (Abcam: ab27156) 14 was used as calibrator(standard curve), and mice treated with control cells (not C1498 cellsAML tumor) were used as a control 13;

FIG. 10E shows flow cytometry analysis of splenocytes isolated on Day 16and stained with anti-CD3-FITC, and anti-CD45-Pacific Blue antibodies,where splenocytes from mice treated with C1498 cells with PBS 12, micetreated with C1498 cells without STAVs 45, and mice treated with C1498containing STAVs 44, mice treated with anti-dsDNA (Abcam: ab27156) 14was used as calibrator (standard curve), and mice treated with controlcells (not C1498 cells AML tumor) were used as a control 13;

FIG. 10F shows flow cytometry analysis of splenocytes isolated on Day 16and stained with anti-CD4-PE and anti-CD3-FITC antibodies, wheresplenocytes from mice treated with C1498 cells with PBS 12, mice treatedwith C1498 cells without STAVs 45, and mice treated with C1498containing STAVs 44, mice treated with anti-dsDNA (Abcam: ab27156) 14was used as calibrator (standard curve), and mice treated with controlcells (not C1498 cells AML tumor) were used as a control 13;

FIG. 10G shows flow cytometry analysis of splenocytes isolated on Day 16and stained with anti-CD8a-PercP anti-CD3-FITC antibodies, wheresplenocytes from mice treated with C1498 cells with PBS 12, mice treatedwith C1498 cells without STAVs 45, and mice treated with C1498containing STAVs 44, mice treated with anti-dsDNA (Abcam: ab27156) 14was used as calibrator (standard curve), and mice treated with controlcells (not C1498 cells AML tumor) were used as a control 13;

FIG. 11A shows immunoblot panels revealing phosphorylation of STING(pSTING) and IRF-3 (pIRF3) 4 hours after transfecting interferonregulatory DNA (ISD) in AML and ATLL (ATLL-84c or JAE) relative tounphosphorylated forms of STING and IRF3, and pTBK, cGAS, and b-Actin(loading control);

FIG. 11B shows the presence of fluorescent FAM labelled STAVs in AMLcells;

FIG. 11C shows the presence of fluorescent FAM labelled STAVs in ATLLcells;

FIG. 11D qRT-PCR analysis of CXC110 in human macrophages 16 hours afterexposure to AML cells transfected with STAVs 46 or not (Mock 19 vs. UVirradiated only 41;

FIG. 11E qRT-PCR analysis of IFNB1 in human macrophages 16 hours afterexposure to AML cells transfected with STAVs 46 or not (Mock 19 vs. UVirradiated only 41;

FIG. 11F qRT-PCR analysis of CXC110 in human macrophages 16 hours afterexposure to ATLL (ATLL-84c or JAE) cells transfected with STAVs 47 ornot (Mock 20 vs. UV irradiated only 42;

FIG. 11G qRT-PCR analysis of IFNB1 in human macrophages 16 hours afterexposure to ATLL (ATLL-84c or JAE) cells transfected with STAVs 47 ornot (Mock 20 vs. UV irradiated only 42;

FIG. 12A shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD3-FITC antibodies fromC57/BL56 mice sacrificed on day 25 (9 days after a first boost), wherethe splenocytes were isolated from the mice i.p. injected with PBS 13,EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAS cells 45, orEL4-cGAS cells transfected with STAV1 45 (the mice were i.p. injected 24hours after the cells were transfected with STAV1 (3 μg/mL) for 3 hoursand UV irradiated (120 mJ/cm for 1 minute) and boosted with the STAV1after 16 days;

FIG. 12B shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD4-PE antibodies fromC57/BL56 mice sacrificed on day 25 (9 days after a first boost), wherethe splenocytes were isolated from the mice i.p. injected with PBS 13,EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAS cells 45, orEL4-cGAS cells transfected with STAV1 45 (the mice were i.p. injected 24hours after the cells were transfected with STAV1 (3 μg/mL) for 3 hoursand UV irradiated (120 mJ/cm for 1 minute) and boosted with the STAV1after 16 days;

FIG. 12C shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD8a-PercP antibodies fromC57/BL56 mice sacrificed on day 25 (9 days after a first boost), wherethe splenocytes were isolated from the mice i.p. injected with PBS 13,EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAS cells 45, orEL4-cGAS cells transfected with STAV1 45 (the mice were i.p. injected 24hours after the cells were transfected with STAV1 (3 μg/mL) for 3 hoursand UV irradiated (120 mJ/cm for 1 minute) and boosted with the STAV1after 16 days;

FIG. 12D shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD45-Pacific Blue antibodiesfrom C57/BL56 mice sacrificed on day 25 (9 days after a first boost),where the splenocytes were isolated from the mice i.p. injected with PBS13, EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAS cells45, or EL4-cGAS cells transfected with STAV1 45 (the mice were i.p.injected 24 hours after the cells were transfected with STAV1 (3 μg/mL)for 3 hours and UV irradiated (120 mJ/cm for 1 minute) and boosted withthe STAV1 after 16 days;

FIG. 12E shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD19-Alexa Fluor 700antibodies from C57/BL56 mice sacrificed on day 25 (9 days after a firstboost), where the splenocytes were isolated from the mice i.p. injectedwith PBS 13, EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAScells 45, or EL4-cGAS cells transfected with STAV1 45 (the mice werei.p. injected 24 hours after the cells were transfected with STAV1 (3μg/mL) for 3 hours and UV irradiated (120 mJ/cm for 1 minute) andboosted with STAV1 after 16 days;

FIG. 12F shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD49b-PE/Cy7 antibodies fromC57/BL56 mice sacrificed on day 25 (9 days after a first boost), wherethe splenocytes were isolated from the mice i.p. injected with PBS 13,EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAS cells 45, orEL4-cGAS cells transfected with STAV1 45 (the mice were i.p. injected 24hours after the cells were transfected with STAV1 (3 μg/mL) for 3 hoursand UV irradiated (120 mJ/cm for 1 minute) and boosted with the STAV1after 16 days;

FIG. 12G shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD11b-FITC antibodies fromC57/BL56 mice sacrificed on day 25 (9 days after a first boost), wherethe splenocytes were isolated from the mice i.p. injected with PBS 13,EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAS cells 45, orEL4-cGAS cells transfected with STAV1 45 (the mice were i.p. injected 24hours after the cells were transfected with STAV1 (3 μg/mL) for 3 hoursand UV irradiated (120 mJ/cm for 1 minute) and boosted with the STAV1after 16 days;

FIG. 13A shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD3-FITC antibodies fromC57/BL56 mice sacrificed on day 12, where the splenocytes were isolatedfrom the mice i.p. injected with C1498 cells (24 hours after the cellswere transfected with STAV1 (3 μg/mL) for 3 hours and UV irradiated (120mJ/cm for 1 minute) 44, or the UV irradiated cells (only) 45, and thePBS control 47 and boosted with the STAV1 after 6 days;

FIG. 13B shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD4-PE antibodies fromC57/BL56 mice sacrificed on day 12, where the splenocytes were isolatedfrom the mice i.p. injected with C1498 cells (24 hours after the cellswere transfected with STAV1 (3 μg/mL) for 3 hours and UV irradiated (120mJ/cm for 1 minute) 44, or the UV irradiated cells (only) 45, and thePBS control 47 and boosted with the STAV1 after 6 days;

FIG. 13C shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD8-PercP antibodies fromC57/BL56 mice sacrificed on day 12, where the splenocytes were isolatedfrom the mice i.p. injected with C1498 cells (24 hours after the cellswere transfected with STAV1 (3 μg/mL) for 3 hours and UV irradiated (120mJ/cm for 1 minute) 44, or the UV irradiated cells (only) 45, and thePBS control 47 and boosted with the STAV1 after 6 days;

FIG. 13D shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD45-Pacific Blue antibodiesfrom C57/BL56 mice sacrificed on day 12, where the splenocytes wereisolated from the mice i.p. injected with C1498 cells (24 hours afterthe cells were transfected with STAV1 (3 μg/mL) for 3 hours and UVirradiated (120 mJ/cm for 1 minute) 44, or the UV irradiated cells(only) 45, and the PBS control 47 and boosted with the STAV1 after 6days;

FIG. 13E shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD19-Alexa Fluor 700antibodies from C57/BL56 mice sacrificed on day 12, where thesplenocytes were isolated from the mice i.p. injected with C1498 cells(24 hours after the cells were transfected with STAV1 (3 μg/mL) for 3hours and UV irradiated (120 mJ/cm for 1 minute) 44, or the UVirradiated cells (only) 45, and the PBS control 47 and boosted with theSTAV1 after 6 days;

FIG. 13F shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD49b-PE/Cy7 antibodies fromC57/BL56 mice sacrificed on day 12, where the splenocytes were isolatedfrom the mice i.p. injected with C1498 cells (24 hours after the cellswere transfected with STAV1 (3 μg/mL) for 3 hours and UV irradiated (120mJ/cm for 1 minute) 44, or the UV irradiated cells (only) 45, and thePBS control 47 and boosted with the STAV1 after 6 days;

FIG. 13G shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD11b-FITC antibodies fromC57/BL56 mice sacrificed on day 12, where the splenocytes were isolatedfrom the mice i.p. injected with C1498 cells (24 hours after the cellswere transfected with STAV1 (3 μg/mL) for 3 hours and UV irradiated (120mJ/cm for 1 minute) 44, or the UV irradiated cells (only) 45, and thePBS control 47 and boosted with the STAV1 after 6 days;

FIG. 13H shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD11c-FITC antibodies fromC57/BL56 mice sacrificed on day 12, where the splenocytes were isolatedfrom the mice i.p. injected with C1498 cells (24 hours after the cellswere transfected with STAV1 (3 μg/mL) for 3 hours and UV irradiated (120mJ/cm for 1 minute) 44, or the UV irradiated cells (only) 45, and thePBS control 47 and boosted with the STAV1 after 6 days;

FIG. 14A shows a schematic representation of the dead cell therapy inmurine ALL model (EL4 cells) for a protocol of four (4) groups differingin the amount of STAVs transfected (PBS control (n=3) 12, 5×10⁶cells/mouse (n=3) 72, 1×10⁶ cells/mouse (n=3) 74, 2×10⁵ cells/mouse)(n=3) 76, of mice I.V. sequentially injected every week with theirradiated T-ALL cells (EL4) using the MaxCyte GT system at 4 μgSTAV/1×10⁶ cells and irradiated by UV (120 mJ/cm for 1 minute);

FIG. 14B shows flow cytometry analysis (performed using LSR-II) of themouse T-ALL cells with anti-CD3-FITC, and anti-CD45-PacificBlue on day38 (10 days after the last injection), where the splenocytes wereisolated and stained with the different fluorescently labeledantibodies, where the PBS control (n=3) is shown as 12, 5×10⁶cells/mouse (n=3) 72, 1×10⁶ cells/mouse (n=3) 74, 2×10⁵ cells/mouse)(n=3) 76;

FIG. 14C shows flow cytometry analysis (performed using LSR-II) of themouse T-ALL cells with anti-CD3-FITC, and anti-CD4-PE on day 38 (10 daysafter the last injection), where the splenocytes were isolated andstained with the different fluorescently labeled antibodies, where thePBS control (n=3) is shown as 12, 5×10⁶ cells/mouse (n=3) 72, 1×10⁶cells/mouse (n=3) 74, 2×10⁵ cells/mouse) (n=3) 76;

FIG. 14D shows flow cytometry analysis (performed using LSR-II) of themouse T-ALL cells with anti-CD3-FITC, and anti-CD8a-PercP on day 38 (10days after the last injection), where splenocytes were isolated/stainedwith the different fluorescently labeled antibodies, where PBS control(n=3) is 12, 5×10⁶ cells/mouse (n=3) 72, 1×10⁶ cells/mouse (n=3) 74,2×10⁵ cells/mouse) (n=3) 76;

FIG. 14E shows flow cytometry analysis (performed using LSR-II) of themouse T-ALL cells with anti-CD19-AlexaFluor 700 on day 38 (10 days afterthe last injection), where the splenocytes were isolated and stainedwith the different fluorescently labeled antibodies, where the PBScontrol (n=3) is shown as 12, 5×10⁶ cells/mouse (n=3) 72, 1×10⁶cells/mouse (n=3) 74, 2×10⁵ cells/mouse) (n=3) 76;

FIG. 15A is a flow diagram showing a treatment protocol for cancerrequiring treatment with a plurality of doses of leukemic cells treatedwith a plurality of STAVs selected from the group consisting of STAV1,STAV2, STAV3, STAV4 and STAV5, according to an embodiment of theinvention;

FIG. 15B is a flow diagram showing a treatment protocol for cancerrequiring treatment with a plurality of doses of leukemic cells treatedwith a plurality of STAVs selected from the group consisting of STAV1,STAV2, STAV3, STAV4 and STAV5 and a treatment with a Dendritic Cellvaccine generated with at least one STAV, according to an embodiment ofthe invention;

FIG. 15C is a flow diagram showing a treatment protocol for cancerrequiring treatment with a plurality of doses of leukemic cells treatedwith a plurality of STAVs selected from the group consisting of STAV1,STAV2, STAV3, STAV4 and STAV5 and a treatment with a plurality ofDendritic Cell vaccines generated with a plurality of STAVs selectedfrom the group consisting of STAV1, STAV2, STAV3, STAV4 and STAV5,according to an embodiment of the invention;

FIG. 15D is a flow diagram showing an alternative treatment protocol forcancer requiring treatment with a plurality of doses of leukemic cellstreated with up to five STAVs comprising the group consisting of STAV1,STAV2, STAV3, STAV4 and STAV5 and a treatment with a plurality ofDendritic Cell vaccines generated with a plurality of STAVs selectedfrom the group consisting of STAV1, STAV2, STAV3, STAV4 and STAV5,according to an embodiment of the invention; and

FIG. 16 is a flow diagram showing a limiting toxicity protocol forrelapsed/refractory aggressive leukemia.

DETAILED DESCRIPTION OF THE INVENTION

Listed below are definitions of various terms used in this application.These definitions apply to the terms as they are used throughout thisspecification and claims, unless otherwise limited in specificinstances, either individually or as part of a larger group.

The transitional term ‘comprising’ is synonymous with ‘including’,‘containing,’ or ‘characterized by’ is inclusive or open-ended and doesnot exclude additional, unrecited elements or method steps. Thetransitional phrase ‘consisting of’ excludes any element, step, oringredient not specified in the claim, but does not exclude additionalcomponents or steps that are unrelated to the invention such asimpurities ordinarily associated with a composition. The transitionalphrase ‘consisting essentially of’ limits the scope of a claim to thespecified materials or steps and those that do not materially affect thebasic and novel characteristic(s) of the claimed invention.

The term ‘cancer’ includes, but is not limited to, the followingcancers: epidermoid Oral: buccal cavity, lip, tongue, mouth, pharynx;Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; Lung:bronchogenic carcinoma (squamous cell or epidermoid, undifferentiatedsmall cell, undifferentiated large cell, adenocarcinoma), alveolar(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus(squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma,lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas(ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoidtumors, vipoma), small bowel or small intestines (adenocarcinoma,lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma,lipoma, neurofibroma, fibroma), large bowel or large intestines(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma), colon, colon-rectum, colorectal, rectum; Genitourinarytract: kidney (adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma,leukemia), bladder and urethra (squamous cell carcinoma, transitionalcell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma),testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages;Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma (serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast;Hematologic: blood (myeloid leukemia (acute and chronic), acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma (malignant lymphoma) hairy cell;lymphoid disorders; Skin: malignant melanoma, basal cell carcinoma,squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis,Thyroid gland: papillary thyroid carcinoma, follicular thyroidcarcinoma; medullary thyroid carcinoma, undifferentiated thyroid cancer,multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma;and adrenal glands: neuroblastoma. Thus, the term ‘cancerous cell’ asprovided herein, includes a cell afflicted by any one of theabove-identified conditions.

The term ‘subject’ as used herein refers to a mammal. A subjecttherefore refers to, for example, dogs, cats, horses, cows, sheep, pigs,guinea pigs, rats, mice, monkeys, apes and the like. Preferably thesubject is a human. When the subject is a human, the subject may bereferred to herein as a patient.

The words ‘treat’, ‘treating’ and ‘treatment’ refer to a method ofalleviating or abating a disease and/or its attendant symptoms.

The words ‘preventing’ and ‘prevent’ describe reducing or eliminatingthe onset of the symptoms or complications of the disease, condition ordisorder.

The terms ‘disease(s)’, ‘disorder(s)’, and ‘condition(s)’ are usedinterchangeably, unless the context clearly dictates otherwise.

The term ‘therapeutically effective amount’ of a compound orpharmaceutical composition of the application, as used herein, means asufficient amount of the compound or pharmaceutical composition so as todecrease the symptoms of a disorder in a subject. As is well understoodin the medical arts a therapeutically effective amount of a compound orpharmaceutical composition of this application will be at a reasonablebenefit/risk ratio applicable to any medical treatment. It will beunderstood, however, that the total daily usage of the compounds andcompositions of the present application will be decided by the attendingphysician within the scope of sound medical judgment. The specificmodulatory (e.g., inhibitory or stimulatory) dose for any particularpatient will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

As used herein, the phrase ‘pharmaceutically acceptable’ refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

As used herein, the term ‘pharmaceutically acceptable salt’ refers tothose salts of the compounds formed by the process of the presentapplication which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977), which is herein expresslyincorporated by reference in its entirety and for all purposes. Thesalts can be prepared in situ during the final isolation andpurification of the compounds of the application, or separately byreacting the free base or acid function with a suitable acid or base.

Examples of pharmaceutically acceptable salts include, but are notlimited to, nontoxic acid addition salts: salts formed with inorganicacids such as hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid and perchloric acid, or with organic acids such as aceticacid, maleic acid, tartaric acid, citric acid, succinic acid or malonicacid. Other pharmaceutically acceptable salts include, but are notlimited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, 7-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and arylsulfonate.

As used herein, the term ‘pharmaceutically acceptable ester’ refers toesters of the compounds formed by the process of the present applicationwhich hydrolyze in vivo and include those that break down readily in thehuman body to leave the parent compound or a salt thereof. Suitableester groups include, for example, those derived from pharmaceuticallyacceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic,cycloalkanoic and alkanedioic acids, in which each alkyl or alkenylmoiety advantageously has not more than 6 carbon atoms. Examples ofparticular esters include, but are not limited to, formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

The term ‘pharmaceutically acceptable prodrugs’ as used herein, refersto those prodrugs of the compounds formed by the process of the presentapplication which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the present application.

‘Prodrug’, as used herein, means a compound which is convertible in vivoby metabolic means (e.g., by hydrolysis) to afford any compounddelineated by the formulae of the instant application. Various forms ofprodrugs are known in the art, for example, as discussed in Bundgaard,(ed.), Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.),Methods in Enzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen,et al., (ed). “Design and Application of Prodrugs, Textbook of DrugDesign and Development, Chapter 5, 113-191 (1991); Bundgaard, et al.,Journal of Drug Deliver Reviews, 8:1-38(1992); Bundgaard, J. ofPharmaceutical Sciences, 77:285 et seq. (1988); Higuchi and Stella(eds.) Prodrugs as Novel Drug Delivery Systems, American ChemicalSociety (1975); and Bernard Testa & Joachim Mayer, “Hydrolysis In DrugAnd Prodrug Metabolism: Chemistry, Biochemistry And Enzymology,” JohnWiley and Sons, Ltd. (2002), which are all herein expressly incorporatedby reference in their entireties and for all purposes.

‘Pharmaceutically acceptable excipient’ means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes an excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A pharmaceutically acceptable excipient asused in the specification and claims includes both one and more than onesuch excipient.

This application also encompasses pharmaceutical compositionscontaining, and methods of treating disorders through administering,pharmaceutically acceptable prodrugs of compounds of the application.For example, compounds of the application having free amino, amido,hydroxy or carboxylic groups can be converted into prodrugs. Prodrugsinclude compounds wherein an amino acid residue, or a polypeptide chainof two or more (e.g., two, three or four) amino acid residues iscovalently joined through an amide or ester bond to a free amino,hydroxy or carboxylic acid group of compounds of the application. Theamino acid residues include but are not limited to the 20 naturallyoccurring amino acids commonly designated by three letter symbols andalso includes 4-hydroxyproline, hydroxylysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,citrulline, homocysteine, homoserine, ornithine and methionine sulfone.Additional types of prodrugs are also encompassed. For instance, freecarboxyl groups can be derivatized as amides or alkyl esters. Freehydroxy groups may be derivatized using groups including but not limitedto hemisuccinates, phosphate esters, dimethylaminoacetates, andphosphoryloxymethyloxy carbonyls, as outlined in Advanced Drug DeliveryReviews, 1996, 19, 1-15, which is herein expressly incorporated byreference in its entirety and for all purposes. Carbamate prodrugs ofhydroxy and amino groups are also included, as are carbonate prodrugs,sulfonate esters and sulfate esters of hydroxy groups. Derivatization ofhydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers wherein theacyl group may be an alkyl ester, optionally substituted with groupsincluding but not limited to ether, amine and carboxylic acidfunctionalities, or where the acyl group is an amino acid ester asdescribed above, are also encompassed. Prodrugs of this type aredescribed in J. Med. Chem. 1996, 39, 10, which is herein expresslyincorporated by reference in its entirety and for all purposes. Freeamines can also be derivatized as amides, sulfonamides orphosphonamides. All of these prodrug moieties may incorporate groupsincluding but not limited to ether, amine and carboxylic acidfunctionalities.

Combinations of substituents and variables envisioned by thisapplication are only those that result in the formation of stablecompounds. The term ‘stable’, as used herein, refers to compounds whichpossess stability sufficient to allow manufacture and which maintainsthe integrity of the compound for a sufficient period of time to beuseful for the purposes detailed herein (e.g., therapeutic orprophylactic administration to a subject).

When any variable (e.g., R₁) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with one or more Rmoieties, then R at each occurrence is selected independently from thedefinition of R. Also, combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compoundswithin a designated atom's normal valency.

In addition, some of the compounds of this application have one or moredouble bonds, or one or more asymmetric centers. Such compounds canoccur as racemates, racemic mixtures, single enantiomers, individualdiastereomers, diastereomeric mixtures, and cis- or trans- or E- orZ-double isomeric forms, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)-, or as(D)- or (L)- for amino acids. When the compounds described hereincontain olefinic double bonds or other centers of geometric asymmetry,and unless specified otherwise, it is intended that the compoundsinclude both E and Z geometric isomers. The configuration of anycarbon-carbon double bond appearing herein is selected for convenienceonly and is not intended to designate a particular configuration unlessthe text so states; thus a carbon-carbon double bond depictedarbitrarily herein as trans may be cis, trans, or a mixture of the twoin any proportion. All such isomeric forms of such compounds areexpressly included in the present application.

‘Isomerism’ means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed ‘stereoisomers’. Stereoisomers that are notmirror images of one another are termed ‘diastereoisomers’, andstereoisomers that are non-superimposable mirror images of each otherare termed ‘enantiomers’ or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a ‘racemic mixture’.

A carbon atom bonded to four non-identical substituents is termed a‘chiral center’.

‘Chiral isomer’ means a compound with at least one chiral center.Compounds with more than one chiral center may exist either as anindividual diastereomer or as a mixture of diastereomers, termed‘diastereomeric mixture’. When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center, e.g., carbon. Absolute configuration refers tothe arrangement in space of the substituents attached to the chiralcenter. The substituents attached to the chiral center underconsideration are ranked in accordance with the Sequence Rule of Cahn,Ingold and Prelog. (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385;errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J.Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81;Cahn, J. Chem. Educ. 1964, 41, 116).

‘Geometric isomer’ means the diastereomers that owe their existence tohindered rotation about double bonds. These configurations aredifferentiated in their names by the prefixes cis and trans, or Z and E,which indicate that the groups are on the same or opposite side of thedouble bond in the molecule according to the Cahn-Ingold-Prelog rules.

Furthermore, the structures and other compounds discussed in thisapplication include all atropic isomers thereof. ‘Atropic isomers’ are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques; ithas been possible to separate mixtures of two atropic isomers in selectcases.

‘Tautomer’ is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solid form,usually one tautomer predominates. In solutions where tautomerization ispossible, a chemical equilibrium of the tautomers will be reached. Theexact ratio of the tautomers depends on several factors, includingtemperature, solvent and pH. The concept of tautomers that areinterconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose. Common tautomeric pairs are:ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomerismin heterocyclic rings (e.g., in nucleobases such as adenine, guanine,thymine and cytosine), amine-enamine and enamine-enamine. The compoundsof this application may also be represented in multiple tautomericforms, in such instances, the application expressly includes alltautomeric forms of the compounds described herein (e.g., alkylation ofa ring system may result in alkylation at multiple sites, theapplication expressly includes all such reaction products).

In the present application, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent application includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like. In the present specification, the structuralformula of the compound represents a certain isomer for convenience insome cases, but the present application includes all isomers, such asgeometrical isomers, optical isomers based on an asymmetrical carbon,stereoisomers, tautomers, and the like.

Additionally, the compounds of the present application, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules.Non-limiting examples of hydrates include monohydrates, dihydrates, etc.Non-limiting examples of solvates include ethanol solvates, acetonesolvates, etc.

‘Solvate’ means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent application includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like.

All documents mentioned herein are incorporated herein by reference. Allpublications and patent documents cited in this application areincorporated by reference for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.By their citation of various references in this document, Applicants donot admit any particular reference is ‘prior art’ to their invention.Embodiments of inventive compositions and methods are illustrated in thefollowing examples.

STING is a cellular innate immune receptor essential for controlling thetranscription of numerous host defense genes, including type I IFN andpro-inflammatory cytokines following the recognition of CDN's oraberrant DNA species in the cytosol of the cell. The source of DNA cancomprise the genome of invading pathogens such as herpes simplex 1 virus(HSV1) or CDNs which are known to be secreted by bacteria such asListeria monocytogenes. That is, STING can directly sense CDNs includingc-di-GMP or c-di-AMP secreted by invading intracellular bacteria,cyclic-GMP-AMP (cGAMP) generated by the cellular synthase, CyclicGMP-AMP synthase (cGAS) following association with cytosolic dsDNAspecies such as microbial DNA, or self-DNA. Generally, the cytosol ofthe cell is free of DNA, since it can aggravate STING-dependent cytokineproduction, an event that can lead to lethal auto-inflammatory disease.For example, self-DNA leaked from the nucleus of cells, following celldivision or following DNA damage, is prevented from activating STINGsignaling by the exonuclease DNase III (Trex1). Consequently, defects inTrex1 function can lead to severe auto-inflammatory diseases due toundigested self-DNA triggering STING activity. In addition, followingthe engulfment of apoptotic cells, phagocyte-dependent DNase II plays acritical role in digesting the DNA from the dead cell, to prevent itfrom activating STING-signaling extrinsically within the phagocyte.

As used herein the phrase ‘STING intracellular pathways’ include IRF-3and NF-kB pathways. In the absence of any contrary indication in thespecification, the use of the term approximately means plus or minus tenpercent, e.g., approximately 200 minutes means 200 plus or minus 20minutes.

Self-DNA leaked from the nucleus of the host cell, following celldivision or even as a consequence of DNA damage can activate STING. Suchself-DNA may be responsible for causing a variety of auto-inflammatorydisease such as Systemic Lupus Erythamatosis (SLE) or Aicardi-GoutieresSyndrome (AGS) and may even be associated with inflammation-associatedcancer. Recent insight into the regulation of STING signaling hasgenerated much needed information relating to the causes of inflammatorydisease, providing new opportunities to develop novel anti-inflammatorycompounds that target this pathway.

The ability of dying cells to activate Antigen Presenting Cells (APCs)is carefully controlled to avoid unwanted inflammatory responses. Forexample, following phagocytosis, regular dying cells do not triggerinflammatory responses which can be required for efficient cytotoxic Tlymphocyte (CTL) priming of the immune system.

Tumor cells presumably mimic these processes to avoid activating APCs.However, dying tumor cells contain exogenous innate immune agonists suchas cytosolic DNA. The cytosolic DNA can potently activate APCs in transthrough extrinsic innate immune, STING-dependent signaling, to generatepotent Cytotoxic T Lymphocyte (CTL) activity. In the absence of STINGagonists, dying cells are ineffectual in the stimulation of APCs intrans. Indeed, cytosolic STING activators, including cytosolic DNA andcyclic dinucleotides (CDNs), constitute cellular danger associatedmolecular patterns (DAMPs) usually only generated by viral infection orfollowing DNA-damage events, that can render tumor cells highlyimmunogenic (i.e., STING activators make a ‘cold’ tumor ‘hot’).

Thus, the efficient eradication of apoptotic cells is designed to avoidinvoking an inflammatory event. Dying cells are generally pooractivators of phagocytes and are immunologically indolent due to thegenomic DNA being degraded by host DNases to prevent the intrinsic andextrinsic activation of STING. Tumor cells mimic this efficient processand avoid activating anti-tumor CTL activity. However, cancer cellscontaining cytosolic dsDNA species, that escape degradation, canpotently stimulate APCs, via extrinsic STING-signaling, to promote thecross-presentation of tumor antigen.

STING is activated by cyclic dinucleotides (CDNs) such as cyclic di-GMPand cyclic-di-AMP secreted by intracellular bacteria followinginfection. Alternatively, STING can be activated by cyclic GMP-AMP(cGAMP) generated by a cellular cGAMP synthase cGAS after associationwith aberrant cytosolic dsDNA species, which can include microbial DNAor self-DNA leaked from the nucleus. Notably, STING signaling has beenshown to be important for facilitating anti-tumor T cell activity.Cytosolic dsDNA species present within a dying tumor cell can activateextrinsic STING signaling in phagocytes likely following associationwith cGAS which can generate CDNs.

The phrase ‘STing Dependent AdjuVants’ or ‘STAVs’ refers to dsDNAoligonucleotides of 70 bp which are innate immune activators of STING.The STAV compositions of the present invention comprise at least onemodification which confers increased or enhanced stability to the STAVs,including, for example, improved resistance to nuclease digestion invivo. In an embodiment, the STAV compositions of the present inventionhave undergone a chemical or biological modification to render them morestable. Exemplary modifications to the STAVs include the modification ofa base, for example, the chemical modification of a base.

The term ‘functional’ as used herein means that the STAV has biologicalactivity to activate STING. The STA compositions of the invention areuseful for the treatment of cancer, inflammation and other disorders.The term ‘therapeutic levels’ refers to levels of STAVs above normalphysiological levels, or the levels in the subject prior toadministration of the STAV composition. As provided herein, thecompositions include a transfer vehicle. As used herein, the term‘transfer vehicle’ includes any of the standard pharmaceutical carriers,diluents, excipients and the like which are generally intended for usein connection with the administration of biologically active agents,including nucleic acids. The compositions and in particular the transfervehicles described herein are capable of delivering STAVs to the targetcell. In embodiments, the transfer vehicle is a lipid nanoparticle.

The term ‘complimentary’ when referring to dsDNA as used herein meanstraditional Watson and Crick complementary, at least approximatelyeighty (80) percent, where approximately in this range means plus orminus twenty (20) percent. The phrase ‘the first strand comprises atleast eighty percent complimentary nucleobases with respect to thesecond strand’ implies that a first strand that contains 76 nucleobasesof which 61 nucleobases are adenine means that the second strandcontains at least 61 nucleobases that are thymine.

The term ‘polymer-conjugated lipid’ means a polymer (for example,polyethylene glycol (PEG), polypropylene glycol, polyvinvylpyrrolidone,poly(N-(2-hydroxypropyl)methacrylamide)s and PEGylated liposomes withdifferent functional groups, including methoxy (OCH₃), amino (NH₂),carboxyl (COOH), and hydroxyl (OH) moieties) conjugated with a lipid.For example PEG can be conjugated with myristoyl diglyceride to generateDMG-PEG 2000. Alternatively, PEG can be conjugated with DSPE a watersoluble derivative of phosphatidylethanolamine with (18:0) stearic acidacyl chains to generate DSPE PEG 2000. PEG conjugated lipids canincorporate various functionalized PEG terminal groups including amine,carboxylic acid, azide, aldehyde, thiol, and hydroxyl moieties. PEGconjugated lipids improve circulation times, drug stability, suitabilityof different routes of administration, and help achieve targeted drugdelivery. In an alternative embodiment of the present invention, abranched polymer (e.g., poly(oligo(ethylene glycol) methyl ethermethacrylate, i.e., poly(tri(ethylene glycol) methyl ether methacrylate,poly(tetra(ethylene glycol) methyl ether methacrylate,poly(penta(ethylene glycol) methyl ether methacrylate,poly(hexa(ethylene glycol) methyl ether methacrylate,poly(hepta(ethylene glycol) methyl ether methacrylate,poly(octa(ethylene glycol) methyl ether methacrylate, poly(noan(ethyleneglycol) methyl ether methacrylate) can be conjugated with lipids.

In addition to PEG conjugated lipids, a ‘sterol’ or an unsaturatedsteroid alcohol, can be used to enhance the stability of the LNP.Sterols can include natural sterols and sterols with unnatural ringjunctions. Sterols can be used to assist the efficiency of introducingthe STAV into the cells. Changing the nature of the sterol component canalso be used to alter the efficiency of introducing the STAV into cells.Natural sterols include cholesterol, cholesterol sulfate, desmosterol,stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol,zymosterol, lathosterol, 14-demethyl-lanosterol,8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, FF-MAS, diosgenin,dehydroepiandrosterone (DHEA) sulfate, DHEA, sitosterol, lanosterol-95,zymostenol, sitostanol, campestanol, campesterol, 7-dehydrodesmosterol,pregnenolone, dihydro T-MAS, delta 5-avenasterol, brassicasterol,dihydro FF-MAS, 24-methylene cholesterol,3B-hydroxy-7-oxo-5-cholestenoic acid, 7α-hydroxy-3-oxo-4-cholestenoicacid, 3B,7α-dihydroxy-5-cholestenoic acid,3B,7B-dihydroxy-5-cholestenoic acid, 3B-hydroxy-5-cholestenoic acid,3-oxo-4-cholestenoic acid, 3B,7α,24S-trihydroxy-5-cholestenoic acid,3B,24S-dihydroxy-5-cholestenoic acid, 3B,7α,25-trihydroxy-5-cholestenoicacid, and 30,25-OH-7-oxo-5-cholestenoic acid.

A ‘phospholipid’ means a molecule with a hydrophilic head group and analiphatic chain linked to an alcohol moiety. The nature of the headgroup, the aliphatic chain and the alcohol can be used to generate awide variety of phospholipids. The aliphatic chain includes saturatedacyl chains, saturated alkyl chains, unsaturated acyl chains,unsaturated alkyl chains, saturated acyl chains with ether bonds,saturated alkyl chains with ester bonds, unsaturated acyl chains withether bonds and unsaturated alkyl chains with ester bonds.Glycerophospholipids and sphingomyelins are phospholipids which differbased on the alcohol moieties. Phospholipids includephosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidic acid, phosphatidylinositol, phosphatidylglycerol,cardiolipin, dipalmitoyl, dimyristoyl, DSPC, dioleoyl, andL-α-phosphatidylcholine. In an embodiment of the present invention, thealcohol in the phospholipid can be a C₃ alcohol. In an alternativeembodiment of the present invention, the phospholipid can include aC₄-C₈ alcohol.

An ‘ionizing lipid’ is a class of lipid molecules which remain neutralat physiological pH, but are protonated under acidic conditions.Ionizing lipids promote endosome escape and reduce toxicity of the LNP.Ionizable lipids include7-[(2-Hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl2-octyldecanoate, DODMA (MBN 305A), DLin-KC2-DMA,(6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (D-Lin-MC3-DMA, or MC3), Heptadecan-9-yl8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102),and [(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate)(ALC-0315).

A ‘cationic lipid’ is a class of lipid molecules that are positivelycharged amphiphiles consisting of three basic chemical functionaldomains: a hydrophilic head, a hydrophobic tail, and a tether betweenthe hydrophilic head and the hydrophobic tail. Cationic lipids includeDOTAP, dimethyldioctadecylammonium bromide,33-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol,dimethyldioctadecylammonium,1,2-dimyristoyl-3-trimethylammonium-propane,1,2-stearoyl-3-trimethylammonium-propane andN-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium.

The term ‘LNP’ means a lipid nanoparticle. A LNP represents a particlemade from lipids (e.g., cationic lipids, non-cationic lipids, conjugatedlipids and/or a sterol that prevents aggregation of the nanoparticle),and a STAV, where the STAV is encapsulated within the lipid (e.g.,Nano-STAVs) (the LNPs used in the Nano-STAVs described herein weresynthesized by Precision Nanosystems, South San Francisco, Calif.94080).

In an embodiment of the present invention, LNP formulations can havefour major components, other than the nucleic acid. In an embodiment ofthe present invention, a LNP comprises a phospholipid, a sterol, anionizable lipid, and a polymer-conjugated lipid. In an alternativeembodiment of the present invention, a LNP comprises a phospholipid, asterol, a cationic lipid, and a polymer-conjugated lipid. In anembodiment of the present invention, the cationic lipid can be DOTAP. Inan embodiment of the present invention, the phospholipid can be DSPC. Inan embodiment of the present invention, the sterol can be cholesterol.In an embodiment of the present invention, the ionizable lipid can beMC3. In an embodiment of the present invention, the polymer conjugatedlipid can be DMG-PEG 2000. In an embodiment of the present invention,DSPC, cholesterol, MC3 and DMG-PEG 2000 can be used to generate the LNPto be combined with STAVB1, STAV2 or STAV3 to generate Nano-STAV1,Nano-STAV2 and Nano-STAV3 respectively, where the diameter of thespherical LNPs can be approximately 88 nm, where approximately means+−10nm. In an embodiment of the present invention, the cholesterol can bebetween 35-45% of the LNP composition. In an embodiment of the presentinvention, the LNP comprises a DSPC, cholesterol, an MC3-like lipid anda PEG-conjugated lipid. The phospholipid and cholesterol promotestability and structural integrity of the LNP. The ionizable lipidpromotes electrostatic interaction with the negatively charged nucleicacids and assists intracellular delivery. The polymer-conjugated lipidimproves solubility of the LNP in serum, and circulation by preventingthe particles from aggregating, while retaining good biocompatibilityand having good tolerance characteristics. In an embodiment of thepresent invention, the Nano-STAVs were composed of 76 bp of dsDNAmodified with ps to block exonuclease activity, encapsulated at anitrogen to phosphate mole ratio of approximately 6 (where approximatelymeans plus or minus one). In an embodiment of the present invention, theNano-STAVs can be approximately 100 nm in size, where approximatelymeans plus or minus ten (10) percent. In an embodiment of the presentinvention, the STAVS are approximately 50% encapsulated in theNano-STAVS. In this range approximately means plus or minus twenty (20)percent. In an alternative embodiment of the present invention, theSTAVS are approximately 75% encapsulated in the Nano-STAVS. In thisrange approximately means plus or minus ten (10) percent. In anotherembodiment of the present invention, the STAVS are at leastapproximately 90% encapsulated in the Nano-STAVS. In this rangeapproximately means plus or minus five (5) percent. In anotheralternative embodiment of the present invention, the STAVS areapproximately 98% encapsulated in the Nano-STAVS. In this rangeapproximately means plus or minus one (1) percent. In an embodiment ofthe present invention, the STAVS are approximately 98% encapsulated inthe Nano-STAVS, at a concentration of dsDNA in the LNP in PBS of 0.2mg/mL. LNP can be extremely useful for systemic applications, as theycan exhibit extended circulation lifetimes following i.v. injection,they can accumulate at distal sites, and they can deliver the STAVs atsites distal to the site of administration.

The methods of the invention provide for optional co-delivery of one ormore unique STAVs to target cells, for example, by combining two uniqueSTAVs into a single transfer vehicle. In an embodiment of the presentinvention, a therapeutic first STAV, and a therapeutic second STAV, canbe formulated in a single transfer vehicle and administered. The presentinvention also contemplates co-delivery and/or co-administration of atherapeutic first STAV and a second STAV to facilitate and/or enhancethe function or delivery of one or both the therapeutic first STAV andthe therapeutic second STAV.

Pharmaceutical compositions including a compound of the presentapplication in free form or in a pharmaceutically acceptable salt formin association with at least one pharmaceutically acceptable carrier ordiluent may be manufactured in a conventional manner by mixing,granulating or coating methods. For example, oral compositions can betablets or gelatin capsules comprising the active ingredient togetherwith a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol,cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearicacid, its magnesium or calcium salt and/or polyethyleneglycol; fortablets also c) binders, e.g., magnesium aluminum silicate, starchpaste, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose and or polyvinylpyrrolidone; if desired d)disintegrants, e.g., starches, agar, alginic acid or its sodium salt, oreffervescent mixtures; and/or e) absorbents, colorants, flavors andsweeteners. Injectable compositions can be aqueous isotonic solutions orsuspensions, and suppositories can be prepared from fatty emulsions orsuspensions. The compositions may be sterilized and/or containadjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressureand/or buffers. In addition, they may also contain other therapeuticallyvaluable substances. Suitable formulations for transdermal applicationsinclude an effective amount of a compound of the present applicationwith a carrier. A carrier may include absorbable pharmacologicallyacceptable solvents to assist passage through the skin of the host. Forexample, transdermal devices may be in the form of a bandage comprisinga backing member, a reservoir containing the compound optionally withcarriers, optionally a rate controlling barrier to deliver the compoundto the skin of the host at a controlled and predetermined rate over aprolonged period of time, and means to secure the device to the skin.Matrix transdermal formulations may also be used. Suitable formulationsfor topical application, e.g., to the skin and eyes, are preferablyaqueous solutions, ointments, creams or gels well-known in the art. Suchmay contain solubilizers, stabilizers, tonicity enhancing agents,buffers and preservatives.

The pharmaceutical compositions of the present application comprise atherapeutically effective amount of a compound of the presentapplication formulated together with one or more pharmaceuticallyacceptable carriers. As used herein, the term ‘pharmaceuticallyacceptable carrier’ means a non-toxic, inert solid, semi-solid or liquidfiller, diluent, encapsulating material or formulation auxiliary of anytype. Some examples of materials which may serve as pharmaceuticallyacceptable carriers include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, or potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, polyacrylates, waxes, polyethylenepolyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose andsucrose; starches such as corn starch and potato starch; cellulose andits derivatives such as sodium carboxymethyl cellulose, ethyl celluloseand cellulose acetate; powdered tragacanth; malt; gelatin; talc;excipients such as cocoa butter and suppository waxes, oils such aspeanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; cornoil and soybean oil; glycols such a propylene glycol or polyethyleneglycol; esters such as ethyl oleate and ethyl laurate, agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water, isotonic saline; Ringer's solution; ethyl alcohol,and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

The pharmaceutical compositions of this application may be administeredto humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), buccally, or as an oral or nasal spray.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous, oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from s.c. or intramuscular injection. Thismay be accomplished by the use of a liquid suspension of crystalline oramorphous material with poor water solubility. The rate of absorption ofthe drug then depends upon its rate of dissolution which, in turn, maydepend upon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally administered drug form is accomplished bydissolving or suspending the drug in an oil vehicle.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisapplication with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid compositions of a similar type may also be employed as fillers insoft and hard filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The active compounds may also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents.

Dosage forms for topical or transdermal administration of a compound ofthis application include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, eye ointments, powders and solutionsare also contemplated as being within the scope of this application.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this application, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisapplication, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

The quantity of active ingredient (e.g., a formulation of the disclosedcompound or salt, hydrate, solvate or isomer thereof) in a unit dose ofcomposition is an effective amount and is varied according to theparticular treatment involved. One skilled in the art will appreciatethat it is sometimes necessary to make routine variations to the dosagedepending on the age and condition of the patient. The dosage will alsodepend on the route of administration. A variety of routes arecontemplated, including oral, pulmonary, rectal, parenteral,transdermal, s.c., i.v., intramuscular, intraperitoneal, inhalational,buccal, sublingual, intrapleural, intrathecal, intranasal, and the like.Dosage forms for the topical or transdermal administration of a compoundof this application include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. In one embodiment, theactive compound is mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives, buffersor propellants that are required.

The pharmaceutical compositions containing active compounds of thepresent application may be manufactured in a manner that is generallyknown, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Techniques for formulation and administration of the disclosed compoundsof the application can be found in Remington: the Science and Practiceof Pharmacy, 19t edition, Mack Publishing Co., Easton, Pa. (1995), whichis herein expressly incorporated by reference in its entirety and forall purposes. In an embodiment, the compounds described herein, and thepharmaceutically acceptable salts thereof, are used in pharmaceuticalpreparations in combination with a pharmaceutically acceptable carrieror diluent. Suitable pharmaceutically acceptable carriers include inertsolid fillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

Therapies based on the use CDNs as anti-tumor agents have been tested.In this scenario, CDNs are directly inoculated into tumors whichplausibly stimulate APC activity to augment anti-tumor T cellsresponses. However, while working robustly in murine tumor models, theCDNs have exhibited little effect in human cancer trials, almostcertainly due to their high turnover rate, in vivo. This has led to thegeneration of non-nucleotide-based STING agonists (small drugs), whichmay be able to escape degradation more effectively. STING signaling inthe context of combined treatment with checkpoint inhibitors found thatthe therapeutic effect of an immune checkpoint inhibitory receptor(CTLA-4) and anti-PD-L1 monoclonal antibodies was lost inSTING-deficient mice. In an embodiment of the present invention, STAVsrepresent a new generation of innate immune activators that triggerSTING signaling.

Retrieved tumor cells transfected with STAVs activate APCs in trans andcan generate potent anti-tumor T cell activity. Immunocompetent micebearing metastatic syngeneic tumors can be treated with STAV ‘loaded’tumor cells after reinfusion and inoculation. Select leukemias, such asacute myeloid leukemia (AML), acute lymphocytic leukemia (ALL) and adultT cell leukemia (ALL) can theoretically be amenable to treatment withSTAVs. Further, the range of cancers can be extended to includemelanomas and cutaneous T cell lymphomas. The i.t. inoculation ofsyngeneic melanoma tumors (B16) in immunocompetent mice can be used togenerate effective anti-tumor CTL activity and cause tumor regression.However, in situations where it is not feasible to retrieve sufficienttumor cells to carry out the transfection with STAVs for re-infusion,the STAV based approach may not be applicable.

In an embodiment of the present invention, the direct introduction ofthe STAVs into the tumor microenvironment (TME) can represent asignificant advance. Further, in various embodiments of the presentinvention, the range of cancers amenable to STAV therapy can be extendedusing a non-cell based LNP strategy that effectively delivers highconcentrations of Nano-STAVs into the TME to potently generateanti-tumor cytotoxic T cell activity. In an embodiment of the presentinvention, the tumor regression generated by Nano-STAVs can be augmentedby co-delivery of checkpoint inhibitors.

In an embodiment of the present invention, data indicates thatNano-STAVs are a potent anti-tumor therapy that suppresses the growth oflocalized tumors (B16 melanoma model in C57/BL6 mice). In an embodimentof the present invention, the tumor regression effect was greatlyaugmented with the synergistic addition of checkpoint inhibitors. In anembodiment of the present invention, the activation of STING signalingin APC's is a main mechanism of generating anti-tumor T cell activityand is capable of overcoming resistance to checkpoint therapy. In anembodiment of the present invention, the benefit of Nano-STAVs oversmall drug agonists is that the procedure mimics the normal process ofantigen cross-presentation, is non-toxic, simple, and inexpensive.

Some of the foregoing compounds can comprise one or more asymmetriccenters, and thus can exist in various isomeric forms, e.g.,stereoisomers and/or diastereomers. Accordingly, compounds of theapplication may be in the form of an individual enantiomer, diastereomeror geometric isomer, or may be in the form of a mixture ofstereoisomers. In one embodiment, the compounds of the application areenantiopure compounds. In another embodiment, mixtures of stereoisomersor diastereomers are provided.

Another aspect is an isotopically labeled compound of any of theformulae delineated herein. Such compounds have one or more isotopeatoms which may or may not be radioactive (e.g., ³H, ²H, ⁴C, ¹³C, ¹⁸F,³⁵S, ³²P, ¹²⁵I, and ¹³¹I) introduced into the compound. Such compoundsare useful for drug metabolism studies and diagnostics, as well astherapeutic applications.

Potency can also be determined by IC₅₀ value. A compound with a lowerIC₅₀ value, as determined under substantially similar conditions, ismore potent relative to a compound with a higher IC₅₀ value. In someembodiments, the substantially similar conditions comprise determiningthe level of binding of a known STING ligand to a STING protein, invitro or in vivo, in the presence of a compound of the application.

In one embodiment, the compounds of the present application are usefulas therapeutic agents, and thus may be useful in the treatment of adisease caused by, or associated with, STING expression, activity,and/or function (e.g., deregulation of STING expression, activity,and/or function) or a disease associated with one or more of theintracellular pathways that STING is involved in (e.g. regulation ofintracellular DNA-mediated type I interferon activation), such as thosedescribed herein.

A ‘selective STING modulator’ can be identified, for example, bycomparing the ability of a compound to modulate STINGexpression/activity/function to its ability to modulate the otherproteins or a STING protein from another species. In some embodiments,the selectivity can be identified by measuring the EC₅₀ or IC₅₀ of thecompounds.

The compounds of the application are defined herein by their chemicalstructures and/or chemical names. Where a compound is referred to byboth a chemical structure and a chemical name, and the chemicalstructure and chemical name conflict, the chemical structure isdeterminative of the compound's identity.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof.

In another aspect, the application provides a method of synthesizing acompound disclosed herein. The synthesis of the compounds of theapplication can be found herein and in the Examples below. Otherembodiments are a method of making a compound of any of the formulaeherein using any one, or combination of, reactions delineated herein.The method can include the use of one or more intermediates or chemicalreagents delineated herein.

The application also provides for a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of theapplication, or a pharmaceutically acceptable salt or ester thereof, anda pharmaceutically acceptable carrier.

Another aspect of the present application relates to a kit comprising acompound of the application or a pharmaceutically acceptable salt orester thereof, or a pharmaceutical composition of the application. Inanother aspect, the application provides a kit comprising a compoundcapable of modulating STING activity selected from one or more compoundsdisclosed herein, or a pharmaceutically acceptable salt or esterthereof, optionally in combination with a second agent and instructionsfor use.

Another aspect of the present application relates to a compound of theapplication or a pharmaceutically acceptable salt or ester thereof, or apharmaceutical composition of the application, for use in themanufacture of a medicament for modulating (e.g., inhibiting orstimulating) a STING protein, for treating or preventing a disease,wherein the diseases is caused by, or associated with, STING expression,activity, and/or function (e.g., deregulation of STING expression,activity, and/or function), or for treating or preventing a diseaseassociated with deregulation of one or more of the intracellularpathways in which a STING protein is involved (e.g., deregulation ofintracellular dsDNA mediated type I interferon activation).

Another aspect of the present application relates to use of a compoundof the application or a pharmaceutically acceptable salt or esterthereof, or a pharmaceutical composition of the application, in themanufacture of a medicament for modulating (e.g., inhibiting orstimulating) a STING protein, for treating or preventing a disease,wherein the diseases is caused by, or associated with, STING expression,activity, and/or function (e.g., deregulation of STING expression,activity, and/or function), or for treating or preventing a diseaseassociated with deregulation of one or more of the intracellularpathways in which a STING protein is involved (e.g., deregulation ofintracellular dsDNA mediated type I interferon activation).

Another aspect of the present application relates to a compound of theapplication or a pharmaceutically acceptable salt or ester thereof, or apharmaceutical composition of the application, for use in modulating(e.g., inhibiting or stimulating) a STING protein, in treating orpreventing a disease, wherein the diseases is caused by, or associatedwith, STING expression, activity, and/or function (e.g., deregulation ofSTING expression, activity, and/or function), or in treating orpreventing a disease associated with deregulation of one or more of theintracellular pathways in which a STING protein is involved (e.g.deregulation of intracellular dsDNA mediated type I interferonactivation).

Another aspect of the present application relates to use of a compoundof the application or a pharmaceutically acceptable salt or esterthereof, or a pharmaceutical composition of the application, inantagonizing a STING protein, in treating or preventing a disease,wherein the diseases is caused by, or associated with, STING expression,activity, and/or function (e.g., deregulation of STING expression,activity, and/or function), or in treating or preventing a diseaseassociated with deregulation of one or more of the intracellularpathways in which a STING protein is involved (e.g., deregulation ofintracellular dsDNA mediated type I interferon activation).

Method of Synthesizing the Compounds

Compounds of the present application can be prepared in a variety ofways using commercially available starting materials, compounds known inthe literature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5^(th) edition, John Wiley & Sons: New York, 2001; andGreene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley & Sons: New York, 1999 are useful andrecognized reference textbooks of organic synthesis known to those inthe art. The following descriptions of synthetic methods are designed toillustrate, but not to limit, general procedures for the preparation ofcompounds of the present application. The processes generally providethe desired final compound at or near the end of the overall process,although it may be desirable in certain instances to further convert thecompound to a pharmaceutically acceptable salt, ester, or prodrugthereof. Suitable synthetic routes are depicted in the schemes below.

Synthesis and Evaluation of STAVs

In an embodiment of the present invention, a variety of ssDNA and dsDNAoligonucleotides, containing exonuclease resistant phosphorothioates atthe ends (ES) that varied in their nucleotide content were synthesized(clinical grade, TriLink Biotechnologies) using procedures known to aperson of ordinary skill in the art. The ssDNA and dsDNAoligonucleotides evaluated to determine which was better at stimulatingSTING signaling following transfection of normal human and mouse cellsincluding APCs had nucleotide content as follows:

A:T30ES = polyA30ES (SEQ ID NO: 1) + polyT30ES (SEQ ID NO: 2)polyA30ES is (SEQ ID NO: 1)A(ps)A(ps)A(ps)AAAAAAAAAAAAAAAAAAAAAAAA(ps)A(ps)A (ps)A, polyT30ES is(SEQ ID NO: 2) T(ps)T(ps)T(ps)TTTTTTTTTTTTTTTTTTTTTTTT(ps)T(ps)T (ps)T.A:T50ES = polyA50ES (SEQ ID NO: 3) + polyT50ES (SEQ ID NO: 4)polyA50ESis (SEQ ID NO: 3)A(ps)A(ps)A(ps)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(ps)A(ps)A(ps)A, polyT50ES is (SEQ ID NO: 4)T(ps)T(ps)T(ps)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT(ps)T(ps)T(ps)T. A:T60ES = polyA60ES (SEQ ID NO: 5) +polyT60ES (SEQ ID NO: 6) polyA60ES is (SEQ ID NO: 5)A(ps)A(ps)A(ps)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(ps)A(ps)A(ps)A, polyT60ES is (SEQ ID NO: 6)T(ps)T(ps)T(ps)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT(ps)T(ps)T(ps)T.A:T70ES = polyA70ES (SEQ ID NO: 7) + polyT70ES (SEQ ID NO: 8)polyA70ES is (SEQ ID NO: 7)A(ps)A(ps)A(ps)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(ps)A(ps)A(ps)A, polyT70ES is(SEQ ID NO: 8) T(ps)T(ps)T(ps)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT(ps)T(ps)T(ps)T.A:T80ES = polyA80ES (SEQ ID NO: 9) + polyT80ES (SEQ ID NO: 10)polyA80ES is (SEQ ID NO: 9)A(ps)A(ps)A(ps)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(ps)A(ps) A(ps)A, polyT80ES is(SEQ ID NO: 10) T(ps)T(ps)T(ps)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT(PS)T(PS) T(PS)T.A:T90ES = polyA90ES (SEQ ID NO: 11) + polyT90ES (SEQ ID NO: 12)polyA90ES is (SEQ ID NO: 11)A(ps)A(ps)A(ps)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA A(ps)A(ps)A(ps)A,polyT90ES is (SEQ ID NO: 12)T(ps)T(ps)T(ps)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT T(PS)T(PS)T(Ps)T.A:T100ES = polyA100ES (SEQ ID NO: 13) + polyT100ES (SEQ ID NO: 14)polyA100ES is (SEQ ID NO: 13)A(ps)A(ps)A(ps)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(ps)A(ps)A(ps)A, polyT100ES is (SEQ ID NO: 14)T(ps)T(ps)T(ps)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT(PS)T(PS)T(PS)T. A:T110ES = polyA110ES (SEQ ID NO: 15)polyT110ES (SEQ ID NO: 16) polyA110ES is (SEQ ID NO: 15)A(ps)A(ps)A(ps)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(ps)A(ps)A(ps)A, polyT110ES (SEQ ID NO: 16)T(ps)T(ps)T(ps)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT(PS)T(ps)T(ps)T. GC30-100ES (SEQ ID NO: 17)GC30ES is G(ps)C(ps)G(ps)CGCGCGCGCGCGCGCGCGCGCGCG (ps)C(ps)G(ps)C.GC50ES is (SEQ ID NO: 18)G(ps)C(ps)G(ps)CGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCG(ps)C(ps)G(ps)C, GC60ES is (SEQ ID NO: 19)G(ps)C(ps)G(ps)CGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCG(ps)C(ps)G(ps)C, GC70ES is (SEQ ID NO: 20)G(ps)C(ps)G(ps)CGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCG(ps)C(ps)G(ps)C, GC80ES is (SEQ ID NO: 21)G(ps)C(ps)G(ps)CGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCG(ps)C(ps) G(ps)C, GC90ES is(SEQ ID NO: 22) G(ps)C(ps)G(ps)CGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGC G(ps)C(ps)G(ps)C, andGC100ES is (SEQ ID NO: 23)G(ps)C(ps)G(ps)CGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCG(ps)C(ps)G(ps)C. STAV1 = polyA76ES (SEQ ID NO: 24) +polyT76ES (SEQ ID NO: 25) PolyA76ES is (SEQ ID NO: 24)A(ps)A(ps)A(ps)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(ps)A(ps)A (ps)A, PolyT76ES(SEQ ID NO: 25) T(ps)T(ps)T(ps)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT(ps)T(ps)T (ps)T.STAV2 = polyAC76ES (SEQ ID NO: 26) polyTG76ES (SEQ ID NO: 27)PolyAC76ES is (SEQ ID NO: 26)A(ps)C(ps)A(ps)CACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACA(ps)C(ps)A (ps)C, PolyTG76ES(SEQ ID NO: 27) G(ps)T(ps)G(ps)TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG(ps)T(ps)G (ps)T. PolyXX76ESPolyAT76ES is (SEQ ID NO: 28)A(ps)T(ps)A(ps)TATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATA(ps)T(ps)A (ps)T, PolyTA76ES is(SEQ ID NO: 29) T(ps)A(ps)T(ps)ATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATAT(ps)A(ps)T (ps)A, PolyACTG76ES is(SEQ ID NO: 30) A(ps)C(ps)T(ps)GACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGA(ps)C(ps)T (ps)G, PolyCAGT76ES is(SEQ ID NO: 31) C(ps)A(ps)G(ps)TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTC(ps)A(ps)G (ps)T, HSVRL2 intron-S is(SEQ ID NO: 32) G(ps)A(ps)C(ps)CCTATCGATACAGGGCACGGGGTCGAACTGTTGGGTTTCGCCATGGTACCCCCTGCATTTATATAGCCAG(ps)A(ps)C (ps)C,HSVRL2 intron-AS is (SEQ ID NO: 33)G(ps)G(ps)T(ps)CTGGCTATATAAATGCAGGGGGTACCATGGCGAAACCCAACAGTTCGACCCCGTGCCCTGTATCGATAGG(ps)G(ps)T (ps)C. PolyX90ESpolyA90ES-FAMisFAM- (SEQ ID NO: 35)A(ps)A(ps)A(ps)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA A(ps)A(ps)A(ps)A,polyT90ES is FAM- (SEQ ID NO: 36)T(ps)T(ps)T(ps)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT T(PS)T(PS)T(Ps)T.STAV3 = polyAT76ES (SEQ ID NO: 37) polyTA76ES (SEQ ID NO: 38)PolyAT:TA76ES is (SEQ ID NO: 37)A(ps)T(ps)T(ps)AATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAA(ps)T(ps)T (ps)A, PolyTA:AT76ES(SEQ ID NO: 38) T(ps)A(ps)A(ps)TTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATTAATT(ps)A(ps)A (ps)T.STAV4 = (SEQ ID NO: 39) + (SEQ ID NO: 40) (SEQ ID NO: 39)A(ps)C(ps)T(ps)GACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGA(ps)C(ps)T (ps)G, (SEQ ID NO: 40)C(ps)A(ps)G(ps)TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTC(ps)A(ps)G (ps)T.STAV5 = (SEQ ID NO: 41) + (SEQ ID NO: 42) (SEQ ID NO: 41)G(ps)A(ps)C(ps)CCTATCGATACAGGGCACGGGGTCGAACTGTTGGGTTTCGCCATGGTACCCCCTGCATTTATATAGCCAG(ps)A(ps)C (ps)C, (SEQ ID NO: 42)G(ps)G(ps)T(ps)CTGGCTATATAAATGCAGGGGGTACCATGGCGAAACCCAACAGTTCGACCCCGTGCCCTGTATCGATAGG(ps)G(ps)T (ps)C.STAV6 = (SEQ ID NO: 43) + (SEQ ID NO: 44) (SEQ ID NO: 43)A(ps)A(ps)A(ps)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA A(ps)A(ps)A(ps)A,(SEQ ID NO: 44) T(ps)T(ps)T(ps)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT T(PS)T(PS)T(PS)T.STAV7 = (SEQ ID NO: 45) + (SEQ ID NO: 46) (SEQ ID NO: 45)G(ps)C(ps)G(ps)CGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGC G(ps)C(ps)G(ps)C,(SEQ ID NO: 46) C(ps)G(ps)C(ps)GCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCG C(ps)G(ps)C(ps)G.

A number of STAVs that were greater than 70 bp were effective instimulating STING-based cytokine production. Based on the result thatSTAVs greater than 70 bp were effective in stimulating STING, three (3)STAVs were used herein as follows: STAV1 a double-stranded polyA:T76ES,oligonucleotides (SEQ ID NO:24)+(SEQ ID NO:25); STAV2 a double-strandedpolyAC:TG76ES, oligonucleotides (SEQ ID NO:26)+(SEQ ID NO:27); and STAV3a double-stranded polyAT:TA76ES, oligonucleotides (SEQ ID NO:37)+(SEQ IDNO:38).

Nano-STAV Synthesis

In an embodiment of the present invention, a LNP can be synthesized fromdistearoylphosphatidylcholine, cholesterol, MC3, and DMG-PEG 2000 bydissolving in ethanol that is rapidly mixed with the STAV1 (SEQ IDNO:24)+(SEQ ID NO:25); STAV2 (SEQ ID NO:26)+(SEQ ID NO:27); or STAV3(SEQ ID NO:37)+(SEQ ID NO:38) in aqueous buffer at a pH approximately 4.The resulting dispersion can then be dialyzed against a normal salinebuffer to remove residual ethanol and raise the pH above approximately7.4, (where approximately means+−pH 1) to produce the finishedNano-STAV1, Nano-STAV2, and Nano-STAV3 respectively.

Check Point Inhibitor Analysis

Anti-PD-L1 (IgG BE0091 or anti-PD-L1 BE0101, BioXcell) and anti-PD1 (J43BE0033-2, BioXcell) were used in the B16 melanoma model. Sex matchedC57/BL6 mice (n=10) were inoculated with B16-OVA (5×10⁵) on the flanks.After 7, 10, and 13 days, when tumors are 50 mm³ in volume, 25 μl (4μg/mL; 0.1 μg/mouse) of Nano-STAVs (STAV1 a double-stranded polyA:T76ES,oligonucleotides (SEQ ID NO:24)+(SEQ ID NO:25); STAV2 a double-strandedpolyAC:TG76ES, oligonucleotides (SEQ ID NO:26)+(SEQ ID NO:27); and STAV3a double-stranded polyAT:TA76ES, oligonucleotides (SEQ ID NO:37)+(SEQ IDNO:38)) were injected i.t. in presence or absence of anti-PD-1 oranti-PD-L1 (50 μg/mouse).

The compounds of the present application can be prepared in a number ofways well known to those skilled in the art of organic synthesis. By wayof example, compounds of the present application can be synthesizedusing the methods described below, together with synthetic methods knownin the art of synthetic organic chemistry, or variations thereon asappreciated by those skilled in the art. Preferred methods include butare not limited to those methods described below.

Compounds of the present application can be synthesized by following thesteps outlined in the following Schemes, which comprise differentsequences of assembling intermediates. Starting materials are eithercommercially available or made by known procedures in the reportedliterature or as illustrated.

A compound of the application can be prepared as a pharmaceuticallyacceptable acid addition salt by reacting the free base form of thecompound with a pharmaceutically acceptable inorganic or organic acid.Alternatively, a pharmaceutically acceptable base addition salt of acompound of the application can be prepared by reacting the free acidform of the compound with a pharmaceutically acceptable inorganic ororganic base. The pharmaceutically acceptable salt may include variouscounterions, e.g., counterions of the inorganic or organic acid,counterions of the inorganic or organic base, or counterions afforded bycounterion exchange.

Acids and bases useful in the methods herein are known in the art. Acidcatalysts are any acidic chemical, which can be inorganic (e.g.,hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic(e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid,ytterbium triflate) in nature. Acids are useful in either catalytic orstoichiometric amounts to facilitate chemical reactions. Bases are anybasic chemical, which can be inorganic (e.g., sodium bicarbonate,potassium hydroxide) or organic (e.g., triethylamine, pyridine) innature. Bases are useful in either catalytic or stoichiometric amountsto facilitate chemical reactions.

Alternatively, the salt forms of the compounds of the application can beprepared using salts of the starting materials or intermediates. Thefree acid or free base forms of the compounds of the application can beprepared from the corresponding base addition salt or acid addition saltfrom, respectively. For example, a compound of the application in anacid addition salt form can be converted to the corresponding free baseby treating with a suitable base (e.g., ammonium hydroxide solution,sodium hydroxide, and the like). A compound of the application in a baseaddition salt form can be converted to the corresponding free acid bytreating with a suitable acid (e.g., hydrochloric acid, etc.).

Those skilled in the art will recognize if a stereo center exists in thecompounds disclosed herein. Accordingly, the present applicationincludes both possible stereoisomers (unless specified in the synthesis)and includes not only racemic compounds but the individual enantiomersand/or diastereomers as well. When a compound is desired as a singleenantiomer or diastereomer, it may be obtained by stereospecificsynthesis or by resolution of the final product or any convenientintermediate. Resolution of the final product, an intermediate, or astarting material may be affected by any suitable method known in theart. See, for example, “Stereochemistry of Organic Compounds” by E. L.Eliel, S. H. Wilen, and L. N. Mander (Wiley-Interscience, 1994).

Compounds of the present application that contain non pyrroloquinoxaline nitrogens can be converted to N-oxides by treatment with anoxidizing agent (e.g., 3-chloroperoxybenzoic acid (m-CPBA) and/orhydrogen peroxides) to afford other compounds of the presentapplication. Thus, all shown and claimed non pyrrolo quinoxalinenitrogen-containing compounds are considered, when allowed by valencyand structure, to include both the compound as shown and its N-oxidederivative (which can be designated as N→O or N⁺—O—). Furthermore, inother instances, the nitrogens in the non pyrrolo quinoxaline compoundsof the present application can be converted to N-hydroxy or N-alkoxycompounds. For example, N-hydroxy compounds can be prepared by oxidationof the parent amine by an oxidizing agent such as m-CPBA. All shown andclaimed non pyrrolo quinoxaline nitrogen-containing compounds are alsoconsidered, when allowed by valency and structure, to cover both thecompound as shown and its N-hydroxy (i.e., N—OH) and N-alkoxy (i.e.,N—OR, wherein R is substituted or unsubstituted C₁-C₆ alkyl, C₁-C₆alkenyl, C₁-C₆ alkynyl, 3-14-membered carbocycle or 3-14-memberedheterocycle) derivatives.

Prodrugs of the compounds of the application can be prepared by methodsknown to those of ordinary skill in the art (e.g., for further detailssee Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters,Vol. 4, p. 1985, which is herein expressly incorporated by reference inits entirety and for all purposes). For example, appropriate prodrugscan be prepared by reacting a non-derivatized compound of theapplication with a suitable carbamylating agent (e.g.,1,1-acyloxyalkylcarbanochloridate, para-nitrophenyl carbonate, or thelike). Specifically, the central N-acetic acid moiety, and otheranalogous carboxylic acid groups, of the compounds of the presentinvention can be modified through techniques known in the art to produceeffective prodrugs of the present invention.

Protected derivatives of the compounds of the application can be made bymeans known to those of ordinary skill in the art. A detaileddescription of techniques applicable to the creation of protectinggroups and their removal can be found in T. W. Greene, “ProtectingGroups in Organic Chemistry”, 3rd edition, John Wiley and Sons, Inc.,1999, which is herein expressly incorporated by reference in itsentirety and for all purposes.

Compounds of the present application can be conveniently prepared, orformed during the process of the application, as solvates (e.g.,hydrates). Hydrates of compounds of the present application can beconveniently prepared by recrystallization from an aqueous/organicsolvent mixture, using organic solvents such as dioxin, tetrahydrofuranor methanol.

Optical isomers may be prepared from their respective optically activeprecursors by the procedures described herein, or by resolving theracemic mixtures. The resolution can be carried out in the presence of aresolving agent, by chromatography or by repeated crystallization or bysome combination of these techniques which are known to those skilled inthe art. Further details regarding resolutions can be found in Jacques,et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons,1981), which is herein expressly incorporated by reference in itsentirety and for all purposes

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. In addition, the solvents, temperatures, reaction durations,etc. delineated herein are for purposes of illustration only and one ofordinary skill in the art will recognize that variation of the reactionconditions can produce the desired bridged macrocyclic products of thepresent application. Synthetic chemistry transformations and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe compounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), which are herein expressly incorporated by reference in theirentireties and for all purposes, and subsequent editions thereof.

In one aspect, the present application provides a method of inhibiting aSTING protein. The method comprises administering to a subject in needthereof an effective amount of a compound of the application or apharmaceutically acceptable salt or ester thereof, or a pharmaceuticalcomposition of the application.

In some embodiments, the modulation of a STING protein activity ismeasured by IC₅₀. In some embodiments, the modulation of a STING proteinactivity is measured by EC₅₀.

A compound of the present application (e.g., a compound of any of theformulae described herein, or selected from any compounds describedherein) is capable of treating or preventing a disease, wherein thediseases is caused by, or associated with, STING expression, activity,and/or function (e.g., deregulation of STING expression, activity,and/or function) or a disease associated with deregulation of one ormore of the intracellular pathways in which a STING protein is involved(e.g., deregulation of intracellular dsDNA mediated type I interferonactivation).

In one aspect, the present application provides a method of treating orpreventing a disease, wherein the diseases is caused by, or associatedwith, STING expression, activity, and/or function (e.g., deregulation ofSTING expression, activity, and/or function). The method comprisesadministering to a subject in need thereof an effective amount of aSTING antagonist compound of the application or a pharmaceuticallyacceptable salt or ester thereof, or a pharmaceutical composition of theapplication. In one aspect, the disease is a STING mediated disorder.

In one aspect, the present application provides a method of treating orpreventing a disease associated with deregulation of one or more of theintracellular pathways in which a STING protein is involved (e.g.,deregulation of intracellular dsDNA mediated type I interferonactivation). The method comprises administering to a subject in needthereof an effective amount of a STING antagonist compound of theapplication or a pharmaceutically acceptable salt or ester thereof, or apharmaceutical composition of the application.

In one embodiment, the present application provides a method of treatingor preventing any of the diseases, disorders, and conditions describedherein, wherein the subject is a human. In one embodiment, theapplication provides a method of treating. In one embodiment, theapplication provides a method of preventing.

As antagonists of a STING protein, the compounds and compositions ofthis application are particularly useful for treating or lessening theseverity of a disease, condition, or disorder where a STING protein orone or more of the intracellular pathways that STING is involved isimplicated in the disease, condition, or disorder. In one embodiment,the present application provides a method for treating or lessening theseverity of a disease, condition, or disorder with STING antagonistcompounds that modulate binding of a cyclic di-nucleotide, (CDN)including non-canonical cyclic di-nucleotide, such as 2′3′cGAMP, to aSTING protein. In one embodiment, the present application provides amethod for treating or lessening the severity of a disease, condition,or disorder with compounds that modulate the synthesis of type Iinterferon and/or type I IFN response and other cytokines, chemokines(STING-inducible proteins).

In one aspect, the present application also provides a method oftreating or preventing cell proliferative disorders such ashyperplasias, dysplasias, or pre-cancerous lesions. Dysplasia is theearliest form of pre-cancerous lesion recognizable in a biopsy by apathologist. The compounds of the present application may beadministered for the purpose of preventing hyperplasias, dysplasias, orpre-cancerous lesions from continuing to expand or from becomingcancerous. Examples of pre-cancerous lesions may occur in skin,esophageal tissue, breast, and cervical intra-epithelial tissue.

In one embodiment, the disease or disorder includes, but is not limitedto, immune disorders, autoimmunity, a cell proliferative disease ordisorder, cancer, inflammation, graft vs host, transplantation,gastrointestinal disorder, rheumatoid arthritis, systemic lupus,cachexia, neurodegenerative disease or disorders, neurological diseasesor disorders, cardiac dysfunction, or microbial infection (e.g., viral,bacterial, and/or fungi infection, parasitic, or infection caused byother microorganism).

In one embodiment, the disease or disorder is a cell proliferativedisease or disorder.

As used herein, the term ‘cell proliferative disorder’ refers toconditions in which unregulated or abnormal growth, or both, of cellscan lead to the development of an unwanted condition or disease, whichmay or may not be cancerous. The term ‘rapidly dividing cell’ as usedherein is defined as any cell that divides at a rate that exceeds or isgreater than what is expected or observed among neighboring orjuxtaposed cells within the same tissue. A cell proliferative disease ordisorder includes a precancer or a precancerous condition. A cellproliferative disease or disorder includes cancer.

In one embodiment, the proliferative disease or disorder isnon-cancerous. In one embodiment, the non-cancerous disease or disorderincludes, but is not limited to, rheumatoid arthritis; inflammation;autoimmune disease; lymphoproliferative conditions; acromegaly;rheumatoid spondylitis; osteoarthritis; gout; other arthriticconditions; sepsis; septic shock; endotoxic shock; gram-negative sepsis;toxic shock syndrome; asthma; adult respiratory distress syndrome;chronic obstructive pulmonary disease; chronic pulmonary inflammation;inflammatory bowel disease; Crohn's disease; skin-relatedhyperproliferative disorders; psoriasis; eczema; atopic dermatitis;hyperpigmentation disorders; eye-related hyperproliferative disorders;age-related macular degeneration; ulcerative colitis; pancreaticfibrosis; hepatic fibrosis; acute and chronic renal disease; irritablebowel syndrome; pyresis; restenosis; cerebral malaria; stroke andischemic injury; neural trauma; Alzheimer's disease; Huntington'sdisease; Parkinson's disease; acute and chronic pain; allergic rhinitis;allergic conjunctivitis; chronic heart failure; acute coronary syndrome;cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter'ssyndrome; acute synovitis; muscle degeneration, bursitis; tendonitis;tenosynovitis; herniated, ruptures, or prolapsed intervertebral disksyndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonarysarcosis; bone resorption diseases, such as osteoporosis;graft-versus-host reaction; fibroadipose hyperplasia; spinocerebullarataxia type 1; CLOVES syndrome; Harlequin ichthyosis; macrodactylysyndrome; Proteus syndrome (Wiedemann syndrome); LEOPARD syndrome;systemic sclerosis; Multiple Sclerosis; lupus; fibromyalgia; AIDS andother viral diseases such as Herpes Zoster, Herpes Simplex I or II,influenza virus and cytomegalovirus; diabetes mellitus;hemihyperplasia-multiple lipomatosis syndrome; megalencephaly; rarehypoglycemia, Klippel-Trenaunay syndrome; harmatoma; Cowden syndrome; orovergrowth-hyperglycemia.

In one embodiment, the proliferative disease or disorder is cancer. Inone embodiment, the cancer is lung cancer, colon cancer, breast cancer,prostate cancer, liver cancer, pancreas cancer, brain cancer, kidneycancer, ovarian cancer, stomach cancer, skin cancer, bone cancer,gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma,hepatocellular carcinoma, papillary renal carcinoma, head and necksquamous cell carcinoma, leukemias, lymphomas, myelomas, or solidtumors.

The term ‘cancer’ includes, but is not limited to, the followingcancers: breast; ovary; cervix; prostate; testis, genitourinary tract;esophagus; larynx, glioblastoma; neuroblastoma; stomach; skin,keratoacanthoma; lung, epidermoid carcinoma, large cell carcinoma, smallcell carcinoma, lung adenocarcinoma; bone; colon; colorectal; adenoma;pancreas, adenocarcinoma; thyroid, follicular carcinoma,undifferentiated carcinoma, papillary carcinoma; seminoma; melanoma;sarcoma; bladder carcinoma; liver carcinoma and biliary passages; kidneycarcinoma; myeloid disorders; lymphoid disorders, Hodgkin's, hairycells; buccal cavity and pharynx (oral), lip, tongue, mouth, pharynx;small intestine; colon, rectum, large intestine, rectum, brain andcentral nervous system; chronic myeloid leukemia (CML), and leukemia.The term ‘cancer’ includes, but is not limited to, the followingcancers: myeloma, lymphoma, or a cancer selected from gastric, renal, orand the following cancers: head and neck, oropharangeal, non-small celllung cancer (NSCLC), endometrial, hepatocarcinoma, Non-Hodgkinslymphoma, and pulmonary.

The term ‘cancer’ also refers to any cancer caused by the proliferationof malignant neoplastic cells, such as tumors, neoplasms, carcinomas,sarcomas, leukemias, lymphomas and the like. For example, cancersinclude, but are not limited to, mesothelioma, leukemias and lymphomassuch as cutaneous T-cell lymphomas (CTCL), noncutaneous peripheralT-cell lymphomas, lymphomas associated with human T-cell lymphotrophicvirus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), B-celllymphoma, acute nonlymphocytic leukemias, chronic lymphocytic leukemia,chronic myelogenous leukemia, acute myelogenous leukemia, lymphomas, andmultiple myeloma, non-Hodgkin lymphoma, acute lymphatic leukemia (ALL),chronic lymphatic leukemia (CLL), Hodgkin's lymphoma, Burkitt lymphoma,adult T-cell leukemia lymphoma, acute-myeloid leukemia (AML), chronicmyeloid leukemia (CML), or hepatocellular carcinoma. Further examplesinclude myelodisplastic syndrome, childhood solid tumors such as braintumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, andsoft-tissue sarcomas, common solid tumors of adults such as head andneck cancers (e.g., oral, laryngeal, nasopharyngeal and esophageal),genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian,testicular), lung cancer (e.g., small-cell and non-small cell), breastcancer, pancreatic cancer, melanoma and other skin cancers, stomachcancer, brain tumors, tumors related to Gorlin's syndrome (e.g.,medulloblastoma, meningioma, etc.), and liver cancer. Additionalexemplary forms of cancer which may be treated by the subject compoundsinclude, but are not limited to, cancer of skeletal or smooth muscle,stomach cancer, cancer of the small intestine, rectum carcinoma, cancerof the salivary gland, endometrial cancer, adrenal cancer, anal cancer,rectal cancer, parathyroid cancer, and pituitary cancer.

Cancer may also include colon carcinoma, familial adenomatous polyposiscarcinoma and hereditary non-polyposis colorectal cancer, or melanoma.Further, cancers include, but are not limited to, labial carcinoma,larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivarygland carcinoma, gastric carcinoma, adenocarcinoma, thyroid cancer(medullary and papillary thyroid carcinoma), renal carcinoma, kidneyparenchyma carcinoma, cervix carcinoma, uterine corpus carcinoma,endometrium carcinoma, chorion carcinoma, testis carcinoma, urinarycarcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma,meningioma, medulloblastoma and peripheral neuroectodermal tumors, gallbladder carcinoma, bronchial carcinoma, multiple myeloma, basalioma,teratoma, retinoblastoma, choroidea melanoma, seminoma,rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma,myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmocytoma.

Cancer may also include colorectal, thyroid, breast, and lung cancer;and myeloproliferative disorders, such as polycythemia vera,thrombocythemia, myeloid metaplasia with myelofibrosis, chronicmyelogenous leukemia, chronic myelomonocytic leukemia, hypereosinophilicsyndrome, juvenile myelomonocytic leukemia, and systemic mast celldisease. In one embodiment, the compounds of this application are usefulfor treating hematopoietic disorders, in particular, acute-myelogenousleukemia (AML), chronic-myelogenous leukemia (CML), acute-promyelocyticleukemia, and acute lymphocytic leukemia (ALL).

Exemplary cancers may also include, but are not limited to,adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma,anal cancer, anorectal cancer, cancer of the anal canal, appendixcancer, childhood cerebellar astrocytoma, childhood cerebralastrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliarycancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer,bladder cancer, uringary bladder cancer, bone and joint cancer,osteosarcoma and malignant fibrous histiocytoma, brain cancer, braintumor, brain stem glioma, cerebellar astrocytoma, cerebralastrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodeimal tumors, visual pathway andhypothalamic glioma, breast cancer, bronchial adenomas/carcinoids,carcinoid tumor, gastrointestinal, nervous system cancer, nervous systemlymphoma, central nervous system cancer, central nervous systemlymphoma, cervical cancer, childhood cancers, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic myeloproliferativedisorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma,lymphoid neoplasm, mycosis fungoides, Seziary Syndrome, endometrialcancer, esophageal cancer, extracranial germ cell tumor, extragonadalgerm cell tumor, extrahepatic bile duct cancer, eye cancer, intraocularmelanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST),germ cell tumor, ovarian germ cell tumor, gestational trophoblastictumor glioma, head and neck cancer, hepatocellular (liver) cancer,Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, ocularcancer, islet cell tumors (endocrine pancreas), Kaposi Sarcoma, kidneycancer, renal cancer, kidney cancer, laryngeal cancer, acutelymphoblastic leukemia, acute myeloid leukemia, chronic lymphocyticleukemia, chronic myelogenous leukemia, hairy cell leukemia, lip andoral cavity cancer, liver cancer, lung cancer, non-small cell lungcancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkinlymphoma, primary central nervous system lymphoma, Waldenstrammacroglobulinemia, medulloblastoma, melanoma, intraocular (eye)melanoma, Merkel cell carcinoma, mesothelioma malignant, mesothelioma,metastatic squamous neck cancer, mouth cancer, cancer of the tongue,multiple endocrine neoplasia syndrome, Mycosis fungoides,myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases,chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma,chronic myeloproliferative disorders, nasopharyngeal cancer,neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer,ovarian cancer, ovarian epithelial cancer, ovarian low malignantpotential tumor, pancreatic cancer, islet cell pancreatic cancer,paranasal sinus and nasal cavity cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor, plasmacell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostatecancer, rectal cancer, renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewingfamily of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterinecancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer(melanoma), merkel cell skin carcinoma, small intestine cancer, softtissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer,supratentorial primitive neuroectodermal tumors, testicular cancer,throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter and otherurinary organs, gestational trophoblastic tumor, urethral cancer,endometrial uterine cancer, uterine sarcoma, uterine corpus cancer,vaginal cancer, vulvar cancer, and Wilm's Tumor.

A ‘cell proliferative disorder of the hematologic system’ is a cellproliferative disease or disorder involving cells of the hematologicsystem. A cell proliferative disorder of the hematologic system caninclude lymphoma, leukemia, myeloid neoplasms, mast cell neoplasms,myelodysplasia, benign monoclonal gammopathy, lymphomatoidgranulomatosis, lymphomatoid papulosis, polycythemia vera, chronicmyelocytic leukemia, agnogenic myeloid metaplasia, and essentialthrombocythemia. A cell proliferative disorder of the hematologic systemcan include hyperplasia, dysplasia, and metaplasia of cells of thehematologic system. Compounds and compositions of the presentapplication may be used to treat a cancer selected from the groupconsisting of a hematologic cancer or a hematologic cell proliferativedisorder. A hematologic cancer can include multiple myeloma, lymphoma(including Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhoodlymphomas, and lymphomas of lymphocytic and cutaneous origin), leukemia(including childhood leukemia, hairy-cell leukemia, acute lymphocyticleukemia, acute myelocytic leukemia, chronic lymphocytic leukemia,chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cellleukemia), myeloid neoplasms, and mast cell neoplasms.

A ‘cell proliferative disorder of the lung’ is a cell proliferativedisease or disorder involving cells of the lung. Cell proliferativedisorders of the lung can include all forms of cell proliferativedisorders affecting lung cells. Cell proliferative disorders of the lungcan include lung cancer, a precancer or precancerous condition of thelung, benign growths or lesions of the lung, and malignant growths orlesions of the lung, and metastatic lesions in tissue and organs in thebody other than the lung. Compounds and compositions of the presentapplication may be used to treat lung cancer or cell proliferativedisorders of the lung. Lung cancer can include all forms of cancer ofthe lung. Lung cancer can include malignant lung neoplasms, carcinoma insitu, typical carcinoid tumors, and atypical carcinoid tumors. Lungcancer can include small cell lung cancer (SCLC), non-small cell lungcancer (NSCLC), squamous cell carcinoma, adenocarcinoma, small cellcarcinoma, large cell carcinoma, adenosquamous cell carcinoma, andmesothelioma. Lung cancer can include ‘scar carcinoma’, bronchioalveolarcarcinoma, giant cell carcinoma, spindle cell carcinoma, and large cellneuroendocrine carcinoma. Lung cancer can include lung neoplasms havinghistologic and ultrastructual heterogeneity (e.g., mixed cell types).

Cell proliferative disorders of the lung can also include hyperplasia,metaplasia, and dysplasia of the lung. Cell proliferative disorders ofthe lung can include asbestos-induced hyperplasia, squamous metaplasia,and benign reactive mesothelial metaplasia. Cell proliferative disordersof the lung can include replacement of columnar epithelium withstratified squamous epithelium, and mucosal dysplasia. Individualsexposed to inhaled injurious environmental agents such as cigarettesmoke and asbestos may be at increased risk for developing cellproliferative disorders of the lung. Prior lung diseases that maypredispose individuals to development of cell proliferative disorders ofthe lung can include chronic interstitial lung disease, necrotizingpulmonary disease, scleroderma, rheumatoid disease, sarcoidosis,interstitial pneumonitis, tuberculosis, repeated pneumonias, idiopathicpulmonary fibrosis, granulomata, asbestosis, fibrosing alveolitis, andHodgkin's disease.

A ‘cell proliferative disorder of the colon’ is a cell proliferativedisorder involving cells of the colon. A cell proliferative disorder ofthe colon includes colon cancer. Compounds and compositions of thepresent application may be used to treat colon cancer or cellproliferative disorders of the colon. Colon cancer can include all formsof cancer of the colon. Colon cancer can include sporadic and hereditarycolon cancers. Colon cancer can include malignant colon neoplasms,carcinoma in situ, typical carcinoid tumors, and atypical carcinoidtumors. Colon cancer can include adenocarcinoma, squamous cellcarcinoma, and adenosquamous cell carcinoma. Colon cancer can beassociated with a hereditary syndrome selected from the group consistingof hereditary nonpolyposis colorectal cancer, familial adenomatouspolyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndromeand juvenile polyposis. Colon cancer can be caused by a hereditarysyndrome selected from the group consisting of hereditary nonpolyposiscolorectal cancer, familial adenomatous polyposis, Gardner's syndrome,Peutz-Jeghers syndrome, Turcot's syndrome, and juvenile polyposis.

Cell proliferative disorders of the colon can also include colon cancer,precancerous conditions of the colon, adenomatous polyps of the colonand metachronous lesions of the colon. A cell proliferative disorder ofthe colon can include adenoma. Cell proliferative disorders of the coloncan be characterized by hyperplasia, metaplasia, and dysplasia of thecolon. Prior colon diseases that may predispose individuals todevelopment of cell proliferative disorders of the colon can includeprior colon cancer. Current disease that may predispose individuals todevelopment of cell proliferative disorders of the colon can includeCrohn's disease and ulcerative colitis. A cell proliferative disorder ofthe colon can be associated with a mutation in a gene selected from thegroup consisting of p53, ras, FAP and DCC. An individual can have anelevated risk of developing a cell proliferative disorder of the colondue to the presence of a mutation in a gene selected from the groupconsisting of p53, ras, FAP and DCC.

A ‘cell proliferative disorder of the pancreas’ is a cell proliferativedisorder involving cells of the pancreas. Compounds and compositions ofthe present application may be used to treat pancreatic cancer or cellproliferative disorders of the pancreas. Cell proliferative disorders ofthe pancreas can include all forms of cell proliferative disordersaffecting pancreatic cells. Cell proliferative disorders of the pancreascan include pancreas cancer, a precancer or precancerous condition ofthe pancreas, hyperplasia of the pancreas, and dysaplasia of thepancreas, benign growths or lesions of the pancreas, and malignantgrowths or lesions of the pancreas, and metastatic lesions in tissue andorgans in the body other than the pancreas. Pancreatic cancer includesall forms of cancer of the pancreas. Pancreatic cancer can includeductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cellcarcinoma, mucinous adenocarcinoma, osteoclast-like giant cellcarcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassifiedlarge cell carcinoma, small cell carcinoma, pancreatoblastoma, papillaryneoplasm, mucinous cystadenoma, papillary cystic neoplasm, and serouscystadenoma. Pancreatic cancer can also include pancreatic neoplasmshaving histologic and ultrastructual heterogeneity (e.g., mixed celltypes).

A ‘cell proliferative disorder of the prostate’ is a cell proliferativedisorder involving cells of the prostate. Compounds and compositions ofthe present application may be used to treat prostate cancer or cellproliferative disorders of the prostate. Cell proliferative disorders ofthe prostate can include all forms of cell proliferative disordersaffecting prostate cells. Cell proliferative disorders of the prostatecan include prostate cancer, a precancer or precancerous condition ofthe prostate, benign growths or lesions of the prostate, and malignantgrowths or lesions of the prostate, and metastatic lesions in tissue andorgans in the body other than the prostate. Cell proliferative disordersof the prostate can include hyperplasia, metaplasia, and dysplasia ofthe prostate.

A ‘cell proliferative disorder of the skin’ is a cell proliferativedisorder involving cells of the skin. Compounds and compositions of thepresent application may be used to treat skin cancer or cellproliferative disorders of the skin. Cell proliferative disorders of theskin can include all forms of cell proliferative disorders affectingskin cells. Cell proliferative disorders of the skin can include aprecancer or precancerous condition of the skin, benign growths orlesions of the skin, melanoma, malignant melanoma and other malignantgrowths or lesions of the skin, and metastatic lesions in tissue andorgans in the body other than the skin. Cell proliferative disorders ofthe skin can include hyperplasia, metaplasia, and dysplasia of the skin.

A ‘cell proliferative disorder of the ovary’ is a cell proliferativedisorder involving cells of the ovary. Compounds and compositions of thepresent application may be used to treat ovarian cancer or cellproliferative disorders of the ovary. Cell proliferative disorders ofthe ovary can include all forms of cell proliferative disordersaffecting cells of the ovary. Cell proliferative disorders of the ovarycan include a precancer or precancerous condition of the ovary, benigngrowths or lesions of the ovary, ovarian cancer, malignant growths orlesions of the ovary, and metastatic lesions in tissue and organs in thebody other than the ovary. Cell proliferative disorders of the skin caninclude hyperplasia, metaplasia, and dysplasia of cells of the ovary.

A ‘cell proliferative disorder of the breast’ is a cell proliferativedisorder involving cells of the breast. Compounds and compositions ofthe present application may be used to treat breast cancer or cellproliferative disorders of the breast. Cell proliferative disorders ofthe breast can include all forms of cell proliferative disordersaffecting breast cells. Cell proliferative disorders of the breast caninclude breast cancer, a precancer or precancerous condition of thebreast, benign growths or lesions of the breast, and malignant growthsor lesions of the breast, and metastatic lesions in tissue and organs inthe body other than the breast. Cell proliferative disorders of thebreast can include hyperplasia, metaplasia, and dysplasia of the breast.

In one embodiment, the disease or disorder includes, but is not limitedto, a disease or disorders caused by or associated with Entamoebahistolytica, Pneumocystis carindi, Trypanosoma cruzi, Trypanosmnabrucei, Leishmania mexicana, Clostridium histolyticum, Staphylococcusaureus, foot-and-mouth disease virus, or Crithidia fasciculata, as wellas disease or disorder associated with osteoporosis, autoimmunity,schistosomiasis, malaria, tumor metastasis, metachromaticleukodystrophy, muscular dystrophy, or amytrophy.

Additional examples of the diseases or disorders include, but are notlimited to, diseases or disorders caused by or associated withveterinary and human pathogenic protozoa, intracellular active parasitesof the phylum Apicomplexa or Sarcomastigophora, Trypanosoma, Plasmodia,Leishmania, Babesia and Theileria, Cryptosporidia, Sacrocystida, Amoeba,Coccidia, and Trichomonadia. For example, the diseases or disordersinclude, but are not limited to, Malaria tropica, caused by, forexample, Plasmodium Alciparum; Malaria terdana, caused by Plasmodiumvivax or Plasmodium ovale, Malaria quartana, caused by Plasmodiummalariae; Toxoplasnosis, caused by Toxoplasma gondii; Coccidiosis,caused for instance by Isospora belli; intestinal Sarcosporidiosis,caused by Sarcocystis suihominis; dysentery caused by Entamoebahistolytica; Cryptosporidiosis, caused by Cryptosporidium parvum;Chagas' disease, caused by Typanosoma cruzi; sleeping sickness, causedby Typanosoma brucei rhodesiense or gambiense, the cutaneous andvisceral as well as other forms of Leishmaniosis; diseases or disorderscaused by veterinary pathogenic protozoa, such as Theileria parva, thepathogen causing bovine East coast fever, Trypanosoma congolensecongolense or Trypanosoma vivax vivax, Trypanosoma brucei brucei,pathogens causing Nagana cattle disease in Africa, Trypanosama bruceievansi causing Surra, Babesia bigemina., the pathogen causing Texasfever in cattle and buffalos, Babesia bovis, the pathogen causingEuropean bovine Babesiosis as well as Babesiosis in dogs, cats andsheep, Sarcocystis ovicanis and ovifelis pathogens causingSarcocystiosis in sheep, cattle and pigs, Cryptosporidia, pathogenscausing Cryptosporidiosis in cattle and birds, Eimeria and Isosporaspecies, pathogens causing Coccidiosis in rabbits, cattle, sheep, goats,pigs and birds, especially in chickens and turkeys. Rickettsia comprisespecies such as Rickettsia felis, Rickettsia prowazekii, Rickettsiaricketti, Rickettsia typhi, Rickettsia conorii, Rickettsia africae andcause diseases such as typhus, rickettsial pox, Boutonneuse fever,African Tick Bite Fever, Rocky Mountain spotted fever, Australian TickTyphus, Flinders Island Spotted Fever and Queensland Tick Typhus.

In one embodiment, the disease or disorder is caused by, or associatedwith, one or more bacteria. Examples of the bacteria include, but arenot limited to, the Gram positive organisms (e.g., Staphylococcusaureus, Staphiococcus epidermidis, Enterococcus faecalis and E. faecium,Streptococcus pneumoniae) and the Gram negative organisms (e.g.,Pseudomonas aeruginosa, Burkholdia cepacia, Xanthomonas nalophila,Escherichia coli, Enterobacter spp, Klebsiella pneumoniae and Salmonellaspp).

In one embodiment, the disease or disorder is caused by, or associatedwith, one or more fungi. Examples of the fingi include, but are notlimited to, Candida albicans, Histoplasma neoformans, Coccidioidesimmitis, and Penicillium marneffei.

In one embodiment, the disease or disorder is a neurological disease ordisorder. In one embodiment, the neurological disease or disorderinvolves the central nervous system (e.g., brain, brainstem andcerebellum), the peripheral nervous system (e.g., cranial nerves),and/or the autonomic nervous system (e.g., parts of which are located inboth central and peripheral nervous system).

Examples of the neurological disorders include, but are not limited to,acquired epileptiform aphasia; acute disseminated encephalomyelitis;adrenoleukodystrophy; age-related macular degeneration; agenesis of thecorpus callosurn; agnosia; Aicardi syndrome; Alexander disease; Alpers'disease; alternating hemiplegia; Alzbeimer's disease; Vascular dementia;amyotrophic lateral sclerosis; anencephaly; Angelman syndrome;angiomatosis; anoxia; aphasia; apraxia; arachnoid cysts; arachnoiditis;Anronl-Chiari malformation; arteriovenous malformation; Aspergersyndrome; ataxia telegiectasia; attention deficit hyperactivitydisorder; autism; autonomic dysfunction; back pain; Batten disease;Behcet's disease; Bell's palsy; benign essential blepharospasm; benignfocal; amyotrophy; benign intracranial hypertension; Binswanger'sdisease; blepharospasm; Bloch Sulzberger syndrome; brachial plexusinjury; brain abscess; brain injury; brain tumors (includingglioblastoma multiforme); spinal tumor; Brown-Sequard syndrome; Canavandisease; carpal tunnel syndrome; causalgia; central pain syndrome;central pontine myelinolysis; cephalic disorder; cerebral aneurysm;cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism;cerebral palsy; Charcot-Marie-Tooth disease; chemotherapy-inducedneuropathy and neuropathic pain; Chiari malformation; chorea; chronicinflammatory demyelinating polyneuropathy; chronic pain; chronicregional pain syndrome; Coffin Lowry syndrome; coma, includingpersistent vegetative state; congenital facial diplegia; corticobasaldegeneration; cranial arteritis; craniosynostosis; Creutzfeldt-Jakobdisease; cumulative trauma disorders; Cushing's syndrome; cytomegalicinclusion body disease; cytomegalovirus infection; dancing eyes-dancingfeet syndrome; Dandy-Walker syndrome; Dawson disease; De Morsier'ssyndrome; Dejerine-Klumke palsy; dementia; dermatomyositis; diabeticneuropathy; diffuse sclerosis; dysautonomia; dysgraphia; dyslexia;dystonias; early infantile epileptic encephalopathy; empty sellasyndrome; encephalitis; encephaloceles; encephalotrigeminalangiomatosis; epilepsy; Erb's palsy; essential tremor; Fabry's disease;Fahr's syndrome; fainting; familial spastic paralysis; febrile seizures;Fisher syndrome; Friedreich's ataxia; fronto-temporal dementia and other‘tauopathies’; Gaucher's disease; Gerstmann's syndrome; giant cellarteritis; giant cell inclusion disease; globoid cell leukodystrophy;Guillain-Barre syndrome; HTLV-1-associated myelopathy;Hallervorden-Spatz disease; head injury; headache; hemifacial spasm;hereditary spastic paraplegia; heredopathia atactica polyneuritiformis;herpes zoster oticus; herpes zoster; Hirayama syndrome; HIV-associateddementia and neuropathy (also neurological manifestations of AIDS);holoprosencephaly; Huntington's disease and other polyglutamine repeatdiseases; hydranencephaly; hydrocephalus; hypercortisolism; hypoxia;immune-mediated encephalomyelitis; inclusion body myositis;incontinentia pigmenti; infantile phytanic acid storage disease;infantile refsum disease; infantile spasms; inflammatory myopathy;intracranial cyst; intracranial hypertension; Joubert syndrome;Kearns-Sayre syndrome; Kennedy disease Kinsbourne syndrome; Klippel Feilsyndrome; Krabbe disease; Kugelberg-Welander disease; kuru; Laforadisease; Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome;lateral medullary (Wallenberg) syndrome; leaning disabilities; Leigh'sdisease; Lennox-Gustaut syndrome; Leseh-Nyhan syndrome; leukodystrophy;Lewy body dementia; Lissencephaly; locked-in syndrome; Lou Giehrig'sdisease (i.e., motor neuron disease or amyotrophic lateral sclerosis);lumbar disc disease; Lyme disease—neurological sequelae; Machado-Josephdisease; macrencephaly; megalencephaly; Melkersson-Rosenthal syndrome;Menieres disease; meningitis; Menkes disease; metachromaticleukodystrophy; microcephaly; migraine; Miller Fisher syndrome;mini-strokes; mitochondrial myopathies; Mobius syndrome; monomelicamyotrophy; motor neuron disease; Moyamoya disease;mucopolysaccharidoses; multi-infarct dementia; multifocal motorneuropathy; multiple sclerosis and other demyelinating disorders;multiple system atrophy with postural hypotension; p muscular dystrophy;myasthenia gravis; myelinoclastic diffuse sclerosis; nyoclonicencephalopathy of infants; myoclonus; myopathy; myotonia congenital;narcolepsy; neurofibromatosis; neuroleptic malignant syndrome;neurological manifestations of AIDS: neurological sequelae of lupus;neuromyotonia; neuronal ceroid lipofuscinosis; neuronal migrationdisorders; Niemann-Pick disease; O'Sullivan-McLeod syndrome; occipitalneuralgia; occult spinal dysraphism sequence; Ohtahara syndrome;olivopontocerebellar atrophy; opsoclonus myoclonus; optic neuritis;orthostatic hypotension; overuse syndrome; paresthesia; Parkinson'sdisease; paramyotonia congenital; paraneoplastic diseases; paroxysmalattacks; Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodicparalyses; peripheral neuropathy; painful neuropathy and neuropathicpain; persistent vegetative state; pervasive developmental disorders;photic sneeze reflex; phytanic acid storage disease; Pick's disease;pinched nerve; pituitary tumors; polymyositis; porencephaly; post-poliosyndrome; postherpetic neuralgia; postinfectious encephalomyelitis;postural hypotension; Prader-Willi syndrome; primary lateral sclerosis;prion diseases; progressive hemifacial atrophy; progressive multifocalleukoencephalopathv; progressive sclerosing poliodystrophy; progressivesupranuclear palsy; pseudotumor cerebri; Ramsay-Hunt syndrome (types Iand II); Rasmussen's encephalitis; reflex sympathetic dystrophysyndrome; Refsum disease; repetitive motion disorders; repetitive stressinjuries; restless legs syndrome; retrovirus-associated myelopathy; Rettsyndrome; Reye's syndrome; Saint Vitus dance; Sandhoff disease;Schilder's disease; schizencephaly; septo-optic dysplasia; shaken babysyndrome; shingles; Shy-Drager syndrome; Sjögren's syndrome; sleepapnea; Soto's syndrome; spasticity; spina bifida; spinal cord injury;spinal cord tumors; spinal muscular atrophy; Stiff-Person syndrome;stroke; Sturge-Weber syndrome; subacute sclerosing panencephalitis;subcortical arteriosclerotic encephalopathy; Sydenham chorea; syncope;syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporalarteritis; tethered spinal cord syndrome; Thomsen disease; thoracicoutlet syndrome; Tic Douloureux; Todd's paralysis; Tourette syndrome;transient ischemic attack; transmissible spongiform encephalopathies;transverse myelitis; traumatic brain injury; tremor; trigeminalneuralgia; tropical spastic paraparesis; tuberous sclerosis; vasculardementia (multi-infarct dementia); vasculitis including temporalarteritis; Von Hippel-Lindau disease; Wallenberg's syndrome;Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome;Wildon's disease; and Zellweger syndrome.

Examples of neurodegenerative diseases may also include, withoutlimitation, Adrenoleukodystrophy (ALD), Alexander's disease, Alper'sdisease, Alzheimer's disease, Amyotrophic lateral sclerosis (LouGehrig's Disease), Ataxia telangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasaldegeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia,Frontotemporal lobar degeneration, Huntington's disease, HIV-associateddementia, Kennedy's disease, Krabbe's disease, Lewy body dementia,Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, NiemannPick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick'sdisease, Primary lateral sclerosis, Prion diseases, ProgressiveSupranuclear Palsy, Refsum's disease, Sandhoff disease, Schilder'sdisease, Subacute combined degeneration of spinal cord secondary toPernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten disease (also knownas Batten disease), Spinocerebellar ataxia (multiple types with varyingcharacteristics), Spinal muscular atrophy, Steele-Richardson-Olszewskidisease, Tabes dorsalis, and Toxic encephalopathy.

In one embodiment, the disease or disorder is an autoimmune disease.Examples of autoimmune diseases include, but are not limited to,rheumatoid arthritis, systemic lupus erythematosus, inflammatory boweldiseases (IBDs) comprising Crohn disease (CD), and ulcerative colitis(UC) which are chronic inflammatory conditions with polygenicsusceptibility.

In one embodiment, the disease or disorder is inflammation, arthritis,rheumatoid arthritis, spondyiarthropathies, gouty arthritis,osteoarthritis, juvenile arthritis, and other arthritic conditions,systemic lupus erthematosus (SLE), skin-related conditions, psoriasis,eczema, burns, dermatitis, neuroinflammation, allergy, pain, neuropathicpain, fever, pulmonary disorders, lung inflammation, adult respiratorydistress syndrome, pulmonary sarcoisosis, asthma, silicosis, chronicpulmonary inflammatory disease, and chronic obstructive pulmonarydisease (COPD), cardiovascular disease, arteriosclerosis, myocardialinfarction (including post-myocardial infarction indications),thrombosis, congestive heart failure, cardiac reperfusion injury, aswell as complications associated with hypertension and/or heart failuresuch as vascular organ damage, restenosis, cardiomyopathy, strokeincluding ischemic and hemorrhagic stroke, reperfusion injury, renalreperfusion injury, ischemia including stroke and brain ischemia, andischemia resulting from cardiac/coronary bypass, neurodegenerativedisorders, liver disease and nephritis, gastrointestinal conditions,inflammatory bowel disease, Crohn's disease, gastritis, irritable bowelsyndrome, ulcerative colitis, ulcerative diseases, gastric ulcers, viraland bacterial infections, sepsis, septic shock, gram negative sepsis,malaria, meningitis, HIV infection, opportunistic infections, cachexiasecondary to infection or malignancy, cachexia secondary to acquiredimmune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex),pneumonia, herpes virus, myalgias due to infection, influenza,autoimmune disease, graft vs. host reaction and allograft rejections,treatment of bone resorption diseases, osteoporosis, multiple sclerosis,cancer, leukemia, lymphoma, colorectal cancer, brain cancer, bonecancer, epithelial call-derived neoplasia (epithelial carcinoma), basalcell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer,mouth cancer, esophageal cancer, small bowel cancer, stomach cancer,colon cancer, liver cancer, bladder cancer, pancreas cancer, ovariancancer, cervical cancer, lung cancer, breast cancer, skin cancer,squamous cell and/or basal cell cancers, prostate cancer, renal cellcarcinoma, and other known cancers that affect epithelial cellsthroughout the body, chronic myelogenous leukemia (CML), acute myeloidleukemia (AML) and acute promyelocytic leukemia (APL), angiogenesisincluding neoplasia, metastasis, central nervous system disorders,central nervous system disorders having an inflammatory or apoptoticcomponent, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, spinal cord injury, peripheralneuropathy, or B-Cell Lymphoma.

In one embodiment, the disease or disorder is selected from autoimmunediseases, inflammatory diseases, proliferative and hyper proliferativediseases, immunologically-mediated diseases, bone diseases, metabolicdiseases, neurological and neurodegenerative diseases, cardiovasculardiseases, hormone related diseases, allergies, asthma, and Alzheimer'sdisease. In one embodiment, the disease or disorder is selected from aproliferative disorder and an immune disorder.

As modulators of a STING protein, the compounds and compositions of thisapplication are also useful in assessing, studying, or testingbiological samples. One aspect of the application relates to modulatingthe activity of a STING protein in a biological sample, comprisingcontacting the biological sample with a compound or a composition of theapplication.

The term ‘biological sample’, as used herein, means an in vitro or an exvivo sample, including, without limitation, cell cultures or extractsthereof; biopsied material obtained from a mammal or extracts thereof;and blood, saliva, urine, feces, semen, tears, or other body fluids orextracts thereof. Modulation (e.g., inhibition or stimulation) ofprotein kinase activity in a biological sample is useful for a varietyof purposes that are known to one of skill in the art. Examples of suchpurposes include, but are not limited to, blood transfusion, organtransplantation, and biological specimen storage.

Another aspect of this application relates to the study of a STINGprotein in biological and pathological phenomena; the study ofintracellular signal transduction pathways mediated by STING protein.Examples of such uses include, but are not limited to, biological assayssuch as enzyme assays and cell-based assays.

The activity of the compounds and compositions of the presentapplication as STING modulators may be assayed in vitro, in vivo, or ina cell line. In vitro assays include assays that determine modulation(e.g., inhibition or stimulation) of binding of a STING ligand to aSTING protein through competitive binding assay. Alternate in vitroassays quantitate the ability of the agonist to bind to the proteinkinase and may be measured either by radio or fluorescent labelling theagonist prior to binding, isolating the ligand/protein complex anddetermining the amount of radio/fluorescent label bound. Detailedconditions for assaying a compound utilized in this application as anantagonist of a STING protein are set forth in the Examples below.

In accordance with the foregoing, the present application provides amethod for preventing or treating any of the diseases or disordersdescribed above in a subject in need of such treatment, comprisingadministering to the subject a therapeutically effective amount of acompound of the application or an enantiomer, diastereomer,stereoisomer, or pharmaceutically acceptable salt thereof, or apharmaceutical composition of the application. For any of the aboveuses, the required dosage will vary depending on the mode ofadministration, the particular condition to be treated and the effectdesired.

Compounds and compositions of the application can be administered intherapeutically effective amounts in a combinational therapy with one ormore therapeutic agents (pharmaceutical combinations) or modalities,e.g., anti-proliferative, anti-cancer, immunomodulatory, oranti-inflammatory agent, and/or non-drug therapies, etc. For example,synergistic effects can occur with anti-proliferative, anti-cancer,immunomodulatory, or anti-inflammatory substances. Where the compoundsof the application are administered in conjunction with other therapies,dosages of the co-administered compounds will of course vary dependingon the type of co-drug employed, on the specific drug employed, on thecondition being treated and so forth.

Combination therapy may include the administration of the subjectcompounds in further combination with one or more other biologicallyactive ingredients (such as, but not limited to, a second STINGmodulator (inhibitor or stimulator), a modulator (inhibitor orstimulator) of the cGAS-CDN-STING axis, or a modulator (inhibitor orstimulator) involved in the intracellular dsDNA mediated type-˜interferon activation. U.S. patent application Ser. No. 16/717,325entitled Modulating Immune Responses inventor Glen N. Barber, filed Dec.17, 2019 is herein incorporated by reference in its entirety and for allpurposes. Other biologically active ingredients may also includeanti-proliferative agents, anti-cancer agents (e.g., chemotherapeuticagents), immunomodulatory agents, antibodies, etc. For instance, thecompounds of the application can be used in combination with otherpharmaceutically active compounds, preferably compounds that are able toenhance the agonist effect of the compounds of the application. Thecompounds of the application can be administered simultaneously (as asingle preparation or separate preparation) or sequentially to the otherdrug therapy or treatment modality. In general, a combination therapyenvisions administration of two or more drugs during a single cycle orcourse of therapy.

In one embodiment, the chemotherapeutic agent is an alkylating agent; anantibiotic; an anti-metabolite; a detoxifying agent; an interferon; apolyclonal or monoclonal antibody; an EGFR inhibitor; a HER2 inhibitor;a histone deacetylase inhibitor; a hormone; a mitotic inhibitor; an MTORinhibitor; a multi-kinase inhibitor; a serine/threonine kinaseinhibitor; a tyrosine kinase inhibitors; a VEGF/VEGFR inhibitor; ataxane or taxane derivative, an aromatase inhibitor, an anthracycline, amicrotubule targeting drug, a topoisomerase poison drug, an inhibitor ofa molecular target or enzyme (e.g., a kinase inhibitor), a cytidineanalog drug, or any chemotherapeutic, anti-neoplastic oranti-proliferative agent listed in www.cancer.org/docroot/cdg/cdg_0.asp,last visited Apr. 27, 2020.

Exemplary alkylating agents include, but are not limited to,cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan(Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU);dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel);ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran);carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide(Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin(Zanosar).

Exemplary antibiotics include, but are not limited to, doxorubicin(Adriamycin); doxorubicin liposomal (Doxil); mitoxantrone (Novantrone);bleomycin (Blenoxane); daunorubicin (Cerubidine); daunorubicin liposomal(DaunoXome); dactinomycin (Cosmegen); epirubicin (Ellence); idarubicin(Idamycin); plicamycin (Mithracin); mitomycin (Mutamycin); pentostatin(Nipent); or valrubicin (Valstar). Exemplary anti-metabolites include,but are not limited to, fluorouracil (Adrucil); capecitabine (Xeloda);hydroxyurea (Hydrea); mercaptopurine (Purinethol); pemetrexed (Alimta);fludarabine (Fludara); nelarabine (Arranon); cladribine (CladribineNovaplus); clofarabine (Clolar); cytarabine (Cytosar-U); decitabine(Dacogen); cytarabine liposomal (DepoCyt); hydroxyurea (Droxia);pralatrexate (Folotyn); floxuridine (FUDR); gemcitabine (Gemzar);cladribine (Leustatin); fludarabine (Oforta); methotrexate (MTX;Rheumatrex); methotrexate (Trexall); thioguanine (Tabloid); TS-1 orcytarabine (Tarabine PFS). Exemplary detoxifying agents include, but arenot limited to, amifostine (Ethyol) or mesna (Mesnex). Exemplaryinterferons include, but are not limited to, interferon alfa-2b (IntronA) or interferon alfa-2a (Roferon-A). Exemplary polyclonal or monoclonalantibodies include, but are not limited to, trastuzumab (Herceptin);ofatumumab (Arzerra); bevacizumab (Avastin); rituximab (Rituxan);cetuximab (Erbitux); panitumumab (Vectibix); tositumomab/iodine³′tositumomab (Bexxar); alemtuzumab (Campath); ibritumomab (Zevalin;In-111; Y-90 Zevalin); gemtuzumab (Mylotarg); eculizumab (Soliris)ordenosumab.

Exemplary EGFR inhibitors include, but are not limited to, gefitinib(Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva);panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab(Emd7200) or EKB-569. Exemplary HER2 inhibitors include, but are notlimited to, trastuzumab (Herceptin); lapatinib (Tykerb) or AC-480.Exemplary histone Deacetylase Inhibitors include, but are not limitedto, vorinostat (Zolinza). Exemplary hormones include, but are notlimited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista);megestrol (Megace); leuprolide (Lupron; Lupron Depot; Eligard; Viadur);fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA;Trelstar Depot); exemestane (Aromasin); goserelin (Zoladex);bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone(Androxy; Halotestin); medroxyprogesterone (Provera; Depo-Provera);estramustine (Emcyt); flutamide (Eulexin); toremifene (Fareston);degarelix (Firmagon); nilutamide (Nilandron); abarelix (Plenaxis); ortestolactone (Teslac).

Exemplary mitotic inhibitors include, but are not limited to, paclitaxel(Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin;Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos;VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole;epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan(Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).Exemplary MTOR inhibitors include, but are not limited to, everolimus(Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus; orAP23573. Exemplary multi-kinase inhibitors include, but are not limitedto, sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474;PKC-412; motesanib; or AP24534. Exemplary serine/threonine kinaseinhibitors include, but are not limited to, ruboxistaurin; eril/easudilhydrochloride; flavopiridol; seliciclib (CYC202; Roscovitrine); SNS-032(BMS-387032); Pkc412; bryostatin; KAI-9803; SF1126; VX-680; Azd1152;Arry-142886 (AZD-6244); SCIO-469; GW681323; CC-401; CEP-1347 or PD332991. Exemplary tyrosine kinase inhibitors include, but are notlimited to, erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec);sorafenib (Nexavar); sunitinib (Sutent); trastuzumab (Herceptin);bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb);cetuximab (Erbitux); panitumumab (Vectibix); everolimus (Afinitor);alemtuzumab (Campath); gemtuzumab (Mylotarg); temsirolimus (Torisel);pazopanib (Votrient); dasatinib (Sprycel); nilotinib (Tasigna);vatalanib (Ptk787; ZK222584); CEP-701; SU5614; MLN518; XL999; VX-322;Azd0530; BMS-354825; SKI-606 CP-690; AG-490; WHI-P154; WHI-P131; AC-220;or AMG888.

Exemplary VEGF/VEGFR inhibitors include, but are not limited to,bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent);ranibizumab; pegaptanib; or vandetinib. Exemplary microtubule targetingdrugs include, but are not limited to, paclitaxel, docetaxel,vincristin, vinblastin, nocodazole, epothilones and navelbine. Exemplarytopoisomerase poison drugs include, but are not limited to, teniposide,etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin,mitoxantrone, amsacrine, epirubicin and idarubicin. Exemplary taxanes ortaxane derivatives include, but are not limited to, paclitaxel anddocetaxol.

Exemplary general chemotherapeutic, anti-neoplastic, anti-proliferativeagents include, but are not limited to, altretamine (Hexalen);isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin(Vesanoid); azacitidine (Vidaza); bortezomib (Velcade) asparaginase(Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine(Matulane); pegaspargase (Oncaspar); denileukin diftitox (Ontak);porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid);bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel);arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine(Leucenol); (1M tegafur—0.4 M 5-chloro-2,4-dihydroxypyrimidine—1 Mpotassium oxonate) or lovastatin.

Exemplary kinase inhibitors include, but are not limited to, Bevacizumab(targets VEGF), BIBW 2992 (targets EGFR and Erb2), Cetuximab/Erbitux(targets Erb1), Imatinib/Gleevic (targets Bcr-Abl), Trastuzumab (targetsErb2), Gefitinib/Iressa (targets EGFR), Ranibizumab (targets VEGF),Pegaptanib (targets VEGF), Erlotinib/Tarceva (targets Erb1), Nilotinib(targets Bcr-Abl), Lapatinib (targets Erb1 and Erb2/Her2),GW-572016/lapatinib ditosylate (targets HER2/Erb2), Panitumumab/Vectibix(targets EGFR), Vandetinib (targets RET/VEGFR), E7080 (multiple targetsincluding RET and VEGFR), Herceptin (targets HER2/Erb2), PKI-166(targets EGFR), Canertinib/CI-1033 (targets EGFR),Sunitinib/SU-11464/Sutent (targets EGFR and FLT3), Matuzumab/Emd7200(targets EGFR), EKB-569 (targets EGFR), Zd6474 (targets EGFR and VEGFR),PKC-412 (targets VEGR and FLT3), Vatalanib/Ptk787/ZK222584 (targetsVEGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN518 (targetsFLT3), XL999 (targets FLT3), VX-322 (targets FLT3), Azd0530 (targetsSRC), BMS-354825 (targets SRC), SKI-606 (targets SRC), CP-690 (targetsJAK), AG-490 (targets JAK), WHI-P154 (targets JAK), WHI-P131 (targetsJAK), sorafenib/Nexavar (targets RAF kinase, VEGFR-1, VEGFR-2, VEGFR-3,PDGFR-β, KIT, FLT-3, and RET), Dasatinib/Sprycel (BCR/ABL and Src),AC-220 (targets Flt3), AC-480 (targets all HER proteins, ‘panHER’),Motesanib diphosphate (targets VEGF1-3, PDGFR, and c-kit), Denosumab(targets RANKL, inhibits SRC), AMG888 (targets HER3), and AP24534(multiple targets including Flt3).

In one embodiment, the compounds may be administered in combination withone or more separate pharmaceutical agents, e.g., a chemotherapeuticagent, an immunotherapeutic agent, or an adjunctive therapeutic agent.

As used herein, ‘combination therapy’ or ‘co-therapy’ includes theadministration of a compound of the present application, or apharmaceutically acceptable salt or ester thereof, and at least a secondagent as part of a specific treatment regimen intended to provide thebeneficial effect from the co-action of these therapeutic agents. Thebeneficial effect of the combination includes, but is not limited to,pharmacokinetic or pharmacodynamic co-action resulting from thecombination of therapeutic agents. Administration of these therapeuticagents in combination typically is carried out over a defined timeperiod (usually minutes, hours, days or weeks depending upon thecombination selected). ‘Combination therapy’ may be, but generally isnot, intended to encompass the administration of two or more of thesetherapeutic agents as part of separate monotherapy regimens thatincidentally and arbitrarily result in the combinations of the presentapplication.

‘Combination therapy’ is intended to embrace administration of thesetherapeutic agents in a sequential manner, wherein each therapeuticagent is administered at a different time, as well as administration ofthese therapeutic agents, or at least two of the therapeutic agents, ina substantially simultaneous manner. Substantially simultaneousadministration can be accomplished, for example, by administering to thesubject a single capsule having a fixed ratio of each therapeutic agentor in multiple, single capsules for each of the therapeutic agents.Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, oral routes, i.v. routes, intramuscular routes, anddirect absorption through mucous membrane tissues. The therapeuticagents can be administered by the same route or by different routes. Forexample, a first therapeutic agent of the combination selected may beadministered by i.v. injection while the other therapeutic agents of thecombination may be administered orally. Alternatively, for example, alltherapeutic agents may be administered orally or all therapeutic agentsmay be administered by i.v. injection. The sequence in which thetherapeutic agents are administered is not narrowly critical.

‘Combination therapy’ also embraces the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies (e.g., surgery orradiation treatment). Where the combination therapy further comprises anon-drug treatment, the non-drug treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and non-drug treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the non-drug treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

The compounds of this application may be modified by appending variousfunctionalities via any synthetic means delineated herein to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological system (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

Check Point Inhibitors

The anti-PD-L1 (IgG BE0091, BE0101 or J43 BE0033-2, BioXcell, NH) andanti-PD1 (CD279, BioXcell, NH) were used in the B16 melanoma model.

Biological Assays

Biological activities of the compounds of the present application can bemeasured by various biochemical or cellular assays known to one ofordinary skill in the art. Non-limiting examples of biochemical andcellular assays are listed in the Examples vide infra.

Pharmaceutical Compositions

In another aspect, a pharmaceutical composition is provided. Thepharmaceutical composition comprises a therapeutically effective amountof a compound of the application, or a pharmaceutically acceptable saltor ester thereof, and a pharmaceutically acceptable carrier.

Compounds of the application may be administered as pharmaceuticalcompositions by any conventional route, in particular internally, e.g.,orally, e.g., in the form of tablets or capsules, or parenterally, e.g.,in the form of injectable solutions or suspensions, or topically, e.g.,in the form of lotions, gels, ointments or creams, or in a nasal orsuppository form.

Example 0

In an embodiment of the present invention, a variety of dsDNA and ssDNAspecies, that varied in their GC or AT content were evaluated todetermine which STAVs were better at stimulating STING signalingfollowing transfection of normal human and mouse cells including APCs.In an embodiment of the present invention, a number of STAVs that weresynthetically generated and contained exonuclease resistantphosphorothioates at the ends (ES). STAVs that were greater than 70 bpwere found to be effective in stimulating STING-based cytokineproduction (FIG. 8 ). FIGS. 8A-F show results for various STING ligandswith different sequences and length. FIG. 8A and FIG. 8B show IFN3 ELISAassay in mouse embryonic fibroblasts (MEFs), hTERT transfected withdifferent lengths of AT rich-STING ligands (A:T30ES (22, A:T50ES (23,A:T60ES (24, A:T70ES (25, A:T80ES (26, A:T90ES (27), and A:T100ES (28).FIG. 8C shows qRT-PCR analysis of IFN31 in human macrophages transfectedwith different length of AT rich-STING ligands. FIG. 8D and FIG. 8E showIFN3 ELISA assays in MEFs, hTERT transfected with different lengths ofGC rich-STING ligands (GC30ES 32, GC50ES 33, GC60ES 34, GC70ES 35,GC80ES 36, GC90ES 37, and GC100ES 38). FIG. 8F shows qRT-PCR analysis ofIFN31 in human macrophages transfected with different length of GCrich-STING ligands (GC30ES 32, GC50ES 33, GC60ES 34, GC70ES 35, GC80ES36, GC90ES 37, and GC100ES 38).

In an embodiment of the present invention, a first STAVs for primaryinoculation (AT rich) can be used and a second STAVs for boostingpurposes (GC) rich can be used to avoid autoimmune targeting of the STAVitself. In an embodiment of the present invention, AT rich STAVs (80 bp)are used. The STAVs were inoculated into tumors (B16-OVA) grown on theflanks of C57/BL6 mice. Unexpectedly, FIGS. 2A-C show significantanti-tumor activity of STAVs in B16 OVA melanoma bearing mice withintact STING signaling resulting in regression of tumors. The mice weresubcutaneously injected with B16-OVA cells on the flank. 10 μg of STAVswere injected i.t. in B16 OVA melanomas every 3 days. The treatment showtumor volumes from WT (n=7/group) from WT (n=4/group) mice injected withSTAV1-STAV5 44 or PBS as control 12 (see FIG. 9A) and STING knock out(SKO) mice (n=7/group) SKO (n=4/group) mice injected with STAV1-STAV5 45or PBS as control 13 (see FIG. 9B) measured on the indicated days. FIG.9C shows the frequency of OVA specific CD8+ T cells in the spleen fromWT (n=4/group) mice injected with STAV1-STAV5 44 or PBS as control 12.FIG. 9C also shows the frequency of OVA specific CD8+ T cells in thespleen from SKO (n=4/group) mice injected with STAV1-STAV5 45 or PBS ascontrol 13. Unexpectedly, the STAV reduces the tumor volume by more thanhalf in the wild type mice with the intact STING gene.

To complement this approach, tumor cells (B16 melanoma) were loaded withpolyA90ES-FAM and polyT90ES-FAM (5′ fluorescently labelled STAVsreferred to as STAVs-FAM (see SEQ ID NO:35, SEQ ID NO:36). The STAV-FAMswere used to visualize the STAVs location in the cells. Greater than 90%of the B16 cells took up the STAVs following transfection (data notshown).

C57/BL6 mice were inoculated with C1498 (murine AML) cells, where theC1498 cells were transfected with STAVs (3 μg/ml) for 3 hours andirradiated by UV (120 mJ/cm for 1 minute) and incubated for 24 hours,followed by sequential intraperitoneal injections of the UV irradiatedAML cells loaded with three (3) distinct STAVs sequences STAV1 on Day 2,STAV2 on Day 5, and STAV3 on Day 10 (FIG. 10 ). Unexpectedly, theSTAVs-based dead cell therapy abolished AML tumor growth as evidenced bymarked reduction in tumor volume and tumor weight as compared to control(PBS) and untreated groups (see FIGS. 10A and 10B). Also unexpectedly,no evidence of antibodies directed against STAV1, STAV2 or STAV3 ordouble stranded DNA were detected from mouse sera (see FIG. 10C), and nonegative effect was observed on CD19+, CD3, CD4, CD8 and CD45 normalimmune cell populations (see FIGS. 10D-10G). STAVs-based dead celltherapy was similarly effective in blocking EL4 (murine T-cell ALLmodel) tumor growth in immunocompetent mice (data not shown).

The integrity of the STING-IRF3 signaling pathway in AML leukemia cells,transfected with interferon stimulatory DNA (ISD) 46 compared with Mock19, and ATLL (ATLL-84c or JAE) leukemia cells, transfected with ISD 47compared with Mock 20, was confirmed to result in phosphorylation(activation) of STING and IRF-3, which is known to be activated by STING(see FIG. 11A for immunoblots revealing phosphorylation of STING(pSTING) and IRF-3 (pIRF3) 4 hours after transfecting with ISD relativeto unphosphorylated forms of STING, IRF3, and pTBK (cGAS, and si-Actin,controls). The presence of fluorescein (FAM) labelled STAVs wasconfirmed in macrophages after phagocytosis of UV irradiated AML andATLL cells transfected with STAVs (FIG. 11B and FIG. 11C). UV irradiatedAML cells loaded with FAM labelled STAVs 46 resulted in robustproduction of CXCL10 in human macrophages (FIG. 11D, Mock 19 and UVirradiated only 41 and of IFNB1 also in human macrophages (FIG. 11E,Mock 19 and UV irradiated only 41. Finally, UV irradiated ATLL cellsloaded with FAM labelled STAVs 47 resulted in robust production ofCXCL10 in human macrophages (FIG. 11F, Mock 20 and UV irradiated only 42and IFNB1 also in human macrophages (FIG. 4G, Mock 20 and UV irradiatedonly 42.

An anti-tumor therapy against re-infusible tumors, such as leukemia bytreating patient's tumors with STAVs, irradiating, and re-infusing. Thetumor cells can be engulfed by APC's and the tumor specific proteinspresented on MHC can prime anti-tumor T cells. EL4 grows in the flanksof C57/BL6 mice or can metastasize to the lungs. EL4-HBZ cells loadedwith STAVs potently activate APCs in a STING-dependent manner.

Using the dead cell immunization protocol, EL4 or EL4-cGAS cells weretransfected with STAV1 (3 μg/mL) for 3 hours and UV irradiated (120mJ/cm for 1 minute) (after 24 hours, C57/BL6 (wild) type mice wereinjected i.p. with the irradiated EL4 or EL4-cGAS cells with/withoutSTAVs, after the primary injection, mice were boosted with theirradiated EL4 or EL4-cGAS cells with/without STAVs at Day 16.

FIG. 12A shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD3-FITC antibodies fromC57/BL56 mice sacrificed on day 25 (9 days after a first boost), wherethe splenocytes were isolated from the mice i.p. injected with PBS 13,EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAS cells 45, orEL4-cGAS cells transfected with STAV1 45 (the mice were i.p. injected 24hours after the cells were transfected with STAV1 (3 μg/mL) for 3 hoursand UV irradiated (120 mJ/cm for 1 minute) and boosted with the STAV1after 16 days.

FIG. 12B shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD4-PE antibodies fromC57/BL56 mice sacrificed on day 25 (9 days after a first boost), wherethe splenocytes were isolated from the mice i.p. injected with PBS 13,EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAS cells 45, orEL4-cGAS cells transfected with STAV1 45 (the mice were i.p. injected 24hours after the cells were transfected with STAV1 (3 μg/mL) for 3 hoursand UV irradiated (120 mJ/cm for 1 minute) and boosted with the STAV1after 16 days.

FIG. 12C shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD8a-PercP antibodies fromC57/BL56 mice sacrificed on day 25 (9 days after a first boost), wherethe splenocytes were isolated from the mice i.p. injected with PBS 13,EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAS cells 45, orEL4-cGAS cells transfected with STAV1 45 (the mice were i.p. injected 24hours after the cells were transfected with STAV1 (3 μg/mL) for 3 hoursand UV irradiated (120 mJ/cm for 1 minute) and boosted with the STAV1after 16 days.

FIG. 12D shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD45-Pacific Blue antibodiesfrom C57/BL56 mice sacrificed on day 25 (9 days after a first boost),where the splenocytes were isolated from the mice i.p. injected with PBS13, EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAS cells45, or EL4-cGAS cells transfected with STAV1 45 (the mice were i.p.injected 24 hours after the cells were transfected with STAV1 (3 μg/mL)for 3 hours and UV irradiated (120 mJ/cm for 1 minute) and boosted withthe STAV1 after 16 days.

FIG. 12E shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD19-Alexa Fluor 700antibodies from C57/BL56 mice sacrificed on day 25 (9 days after a firstboost), where the splenocytes were isolated from the mice i.p. injectedwith PBS 13, EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAScells 45, or EL4-cGAS cells transfected with STAV1 45 (the mice werei.p. injected 24 hours after the cells were transfected with STAV1 (3μg/mL) for 3 hours and UV irradiated (120 mJ/cm for 1 minute) andboosted with the STAV1 after 16 days.

FIG. 12F shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD49b-PE/Cy7 antibodies fromC57/BL56 mice sacrificed on day 25 (9 days after a first boost), wherethe splenocytes were isolated from the mice i.p. injected with PBS 13,EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAS cells 45, orEL4-cGAS cells transfected with STAV1 45 (the mice were i.p. injected 24hours after the cells were transfected with STAV1 (3 μg/mL) for 3 hoursand UV irradiated (120 mJ/cm for 1 minute) and boosted with the STAV1after 16 days.

FIG. 12G shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD11b-FITC antibodies fromC57/BL56 mice sacrificed on day 25 (9 days after a first boost), wherethe splenocytes were isolated from the mice i.p. injected with PBS 13,EL4 cells 12, EL4 cells transfected with STAV1 47, EL4-cGAS cells 45, orEL4-cGAS cells transfected with STAV1 45 (the mice were i.p. injected 24hours after the cells were transfected with STAV1 (3 μg/mL) for 3 hoursand UV irradiated (120 mJ/cm for 1 minute) and boosted with the STAV1after 16 days.

FIG. 13A shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD3-FITC antibodies fromC57/BL56 mice sacrificed on day 12, where the splenocytes were isolatedfrom the mice i.p. injected with C1498 cells (24 hours after the cellswere transfected with STAV1 (3 μg/mL) for 3 hours and UV irradiated (120mJ/cm for 1 minute) 44, or the UV irradiated cells (only) 45, and thePBS control 47 and boosted with the STAV1 after 6 days.

FIG. 13B shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD4-PE antibodies fromC57/BL56 mice sacrificed on day 12, where the splenocytes were isolatedfrom the mice i.p. injected with C1498 cells (24 hours after the cellswere transfected with STAV1 (3 μg/mL) for 3 hours and UV irradiated (120mJ/cm for 1 minute) 44, or the UV irradiated cells (only) 45, and thePBS control 47 and boosted with the STAV1 after 6 days.

FIG. 13C shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD8-PercP antibodies fromC57/BL56 mice sacrificed on day 12, where the splenocytes were isolatedfrom the mice i.p. injected with C1498 cells (24 hours after the cellswere transfected with STAV1 (3 μg/mL) for 3 hours and UV irradiated (120mJ/cm for 1 minute) 44, or the UV irradiated cells (only) 45, and thePBS control 47 and boosted with the STAV1 after 6 days.

FIG. 13D shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD45-Pacific Blue antibodiesfrom C57/BL56 mice sacrificed on day 12, where the splenocytes wereisolated from the mice i.p. injected with C1498 cells (24 hours afterthe cells were transfected with STAV1 (3 μg/mL) for 3 hours and UVirradiated (120 mJ/cm for 1 minute) 44, or the UV irradiated cells(only) 45, and the PBS control 47 and boosted with the STAV1 after 6days.

FIG. 13E shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD19-Alexa Fluor 700antibodies from C57/BL56 mice sacrificed on day 12, where thesplenocytes were isolated from the mice i.p. injected with C1498 cells(24 hours after the cells were transfected with STAV1 (3 μg/mL) for 3hours and UV irradiated (120 mJ/cm for 1 minute) 44, or the UVirradiated cells (only) 45, and the PBS control 47 and boosted with theSTAV1 after 6 days.

FIG. 13F shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD49b-PE/Cy7 antibodies fromC57/BL56 mice sacrificed on day 12, where the splenocytes were isolatedfrom the mice i.p. injected with C1498 cells (24 hours after the cellswere transfected with STAV1 (3 μg/mL) for 3 hours and UV irradiated (120mJ/cm for 1 minute) 44, or the UV irradiated cells (only) 45, and thePBS control 47 and boosted with the STAV1 after 6 days.

FIG. 13G shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD11b-FITC antibodies fromC57/BL56 mice sacrificed on day 12, where the splenocytes were isolatedfrom the mice i.p. injected with C1498 cells (24 hours after the cellswere transfected with STAV1 (3 μg/mL) for 3 hours and UV irradiated (120mJ/cm for 1 minute) 44, or the UV irradiated cells (only) 45, and thePBS control 47 and boosted with the STAV1 after 6 days.

FIG. 13H shows flow cytometry analysis of splenocytes isolated andstained with the fluorescently labeled anti-CD11c-FITC antibodies fromC57/BL56 mice sacrificed on day 12, where the splenocytes were isolatedfrom the mice i.p. injected with C1498 cells (24 hours after the cellswere transfected with STAV1 (3 μg/mL) for 3 hours and UV irradiated (120mJ/cm for 1 minute) 44, or the UV irradiated cells (only) 45, and thePBS control 47 and boosted with the STAV1 after 6 days. Only cellscarrying STAVs were able to therapeutically impair the growth of tumors(see FIG. 13A-H).

Mouse T-ALL cells (EL4) were transfected with STAVs using the MaxCyte GTsystem at 4 μg/1×10⁶ cells and irradiated by UV (120 mJ/cm for 1minute). Mice were i.v. sequentially injected every week with theirradiated T-ALL cells containing STAV1, STAV2, STAV3, STAV4, and STAV5,respectively. Group 1: PBS control (n=3) 12, Group 2: 5×10⁶ cells/mouse(n=3) 72, Group 3: 1×10⁶ cells/mouse (n=3) 74, Group 4: 0.2×10⁶cells/mouse) (n=3) 76. On day 10 after the last injection, splenocyteswere isolated and stained with different fluorescently labeledantibodies (Biolegend: anti-CD3-FITC, anti-CD4-PE, anti-CD8a-PercP,anti-CD45-PacificBlue, anti-CD19-AlexaFluor 700). Flow cytometryanalysis was performed using LSR-II. Collectively, the data suggest noauto-immune responses to the therapy. These experiments were replicatedusing sequential administration of STAVs in the EL4 model and the sameresults were obtained demonstrating that only EL4 cells carrying STAVsimpaired tumor growth (see FIG. 14 ).

FIG. 14A shows a flow diagram for a protocol of 4 groups (12, 72, 74,and 76), of mice were I.V. sequentially injected every week with theirradiated T-ALL cells (EL4) transfected (72, 74, and 76 with STAV1,STAV2, STAV3, STAV4, and STAV5) using the MaxCyte GT system at 4 μgSTAV/1×10⁶ cells and irradiated by UV (120 mJ/cm for 1 minute), wherethe 4 groups were the PBS control (n=3) 12, 5×10⁶ cells/mouse (n=3) 72,1×10⁶ cells/mouse (n=3) 74, 2×10⁵ cells/mouse) (n=3) 76. FIG. 14B showsflow cytometry analysis (performed using LSR-II) of the mouse T-ALLcells with anti-CD3-FITC, and anti-CD45-PacificBlue, where the PBScontrol (n=3) is shown as 12, 5×10⁶ cells/mouse (n=3) 72, 1×10⁶cells/mouse (n=3) 74, 2×10⁵ cells/mouse) (n=3) 76. FIG. 14C shows flowcytometry analysis (performed using LSR-II) of the mouse T-ALL cellswith anti-CD3-FITC, and anti-CD4-PE, where the PBS control (n=3) isshown as 12, 5×10⁶ cells/mouse (n=3) 72, 1×10⁶ cells/mouse (n=3) 74,2×10⁵ cells/mouse) (n=3) 76. FIG. 14D shows flow cytometry analysis(performed using LSR-II) of the mouse T-ALL cells with anti-CD3-FITC,and anti-CD8a-PercP, where the PBS control (n=3) is shown as 12, 5×10⁶cells/mouse (n=3) 72, 1×10⁶ cells/mouse (n=3) 74, 2×10⁵ cells/mouse)(n=3) 76. FIG. 14E shows flow cytometry analysis (performed usingLSR-II) of the mouse T-ALL cells with anti-CD19-AlexaFluor 700, wherethe PBS control (n=3) is shown as 12, 5×10⁶ cells/mouse (n=3) 72, 1×10⁶cells/mouse (n=3) 74, 2×10⁵ cells/mouse) (n=3) 76. On day 38 (10 daysafter the last injection), splenocytes were isolated and stained withthe different fluorescently labeled antibodies.

Mouse Ex Vivo Experiments

Day 1: Subjects can undergo leukapheresis in order to obtain 200-300 mLplasma fraction enriched with peripheral blood mononuclear cells (PBMCs)for purification of leukemic cells (target yield 2.4×10⁹ cells) andmonocytes (target yield 5-30×10⁹ cells).

Transfection of autologous leukemic cells loaded with STAVs. Leukemiccells can be separately transfected (loaded) ex vivo with STAV1, STAV2,STAV3, STAV4, and STAV5, followed by UV irradiation and infused backinto subjects on Days 3, 17, 31, 45, and 59, respectively. Five distinctSTAVs sequences are shown below (synthesized by Trilink Biotechnologies,HPLC Purified, Endotoxin Tested (<5 EU/mL).

DNA vaccine: Mice were immunized with a plasmid encoding OVA by i.m.electroporation (100 μg per mouse). The booster immunization was givenby i.m. two (2) to four (4) weeks after the primary immunization. STINGdeficient animals (−/−) or controls (+/+) have been twice immunizedtwice using i.m. electroporation with a DNA vaccine encoding ovalbumin.Serum was measured for anti-OVA IgG. To evaluate if STING played a rolein this signaling pathway, STING −/− or control mice were immunized withplasmid DNA encoding the ovalbumin gene. While normal B and T cellsubsets were noted in unstimulated STING −/− animals, followingimmunization Sting −/− mice exhibited significantly less serum ovalbumin(OVA) specific immunoglobulin (Ig)G's compared to controls. In addition,spleen CD8 T-cell frequency and IFN-γ secretion was markedly reduced inSting mice following immunization, compared to wild type mice. Sinceimmunoglobulin responses to OVA peptide were normal, these dataemphasized that the STING-governed DNA sensor pathway is essential forefficient DNA vaccine-induced T-cell responses to antigen. Given thisinformation, it was evaluated whether STING played a role infacilitating T-cell responses following infection with the DNA virusvaccinia that expresses ovalbumin (VV-OVA). This study indicated thatcontrol mice, but not Sting (−/−) mice elicited strong T-cell responsesto viral encoded OVA, verifying the importance of STING in innate immunesignaling processes required for DNA adjuvant activity.

STING also appears important for recognizing DNA's ability to stimulatethe innate immune response, including DNA comprising vectors, plasmids,poly dA-dT, poly dC-dG and DNA of varying lengths and sequencecomposition including ISD. Thus, in another preferred embodiment, STINGmodulates the innate immune response. It is concluded that STING mayplay a more predominant role in facilitating RIG-1 mediated innatesignaling rather than MDA5. Interestingly, a significant defect was notdetected (>5-fold) in the ability of transfected B-form DNA, i.e., polydA-dT or non CpG containing ISD to induce IFN3 in MEFs lacking STINGcompared to controls.

Human Ex Vivo Experiments

The proposed treatment consists of combination of STAVs loadedautologous leukemic cells (up to 5 doses) plus syngeneic STAVs augmentedDC cells (up to 4 doses). Treatment-limiting toxicity (TLT) will beassessed over a period of first 60 days, where patients are planned toreceive 9 vaccine doses—5 doses of STAVs loaded cells (Days 3, 17, 31,45, and 59) and 4 doses of DC vaccinations (Days 10, 17, 24, 31).

Eligible subjects with one of the following incurablerelapsed/refractory aggressive leukemias: HTLV-1 associated adult T-cellleukemia-lymphoma (ATLL), Acute myelogenous leukemia (AML), and Acutelymphoblastic leukemia (ALL).

For subjects 2 and 3 enrolled of each cohort (ATLL, AML, ALL), eachsubject will receive dead UV-irradiated STAVs loaded autologous leukemiccells and DC vaccine administration no less than 60 days after the priorenrolled subject 1 has received the second DC vaccination and had noTLTs. After the third subject of each cohort (ATLL, AML, ALL) completes60-day DLT free observation period, no further staggering is requiredfor that specific disease.

If there are 2 TLTs in the first 3 subjects for each cohort (ATLL, AML,ALL), then study accrual will be held until the protocol is re-evaluatedfor safety. If two or more subjects cannot have DC vaccine made fortechnical reasons, then protocol accrual will be held and the protocoland the procedures for manufacture will be evaluated. If subjectsprogress at any time after receiving the second DC but before they areable to receive further complete treatment as planned on study, thenthey will be considered evaluable for TLT. If they progress before thesecond DC administration then they will be replaced for TLT.

The poor prognosis of patients with relapsed/refractory acuteleukemia-lymphomas for which no other effective therapies exits is suchthat it is reasonable to include these patients on an investigationalprotocol using dead STAVs loaded autologous leukemic cells which appearto be safe in pre-clinical animal models, particularly with DCvaccination, which has been associated with few significant toxicitiesin human trials. Because completion of study therapy for a given subjectcan last up to 2 months, in this setting of prolonged treatment it isreasonable to stagger enrollment in the fashion we have described.

STAVs to treat leukemia cells ex vivo followed by re-infusion withautologous DCs stimulated by irradiated autologous UV irradiated (dead)leukemia cells loaded with STAVs. Open-label, phase I study using STAVsto treat leukemia cells ex vivo followed by re-infusion with autologousUV-irradiated (dead) DCs stimulated by irradiated autologous leukemiacells loaded with STAVs.

Day 1: Subjects can undergo leukapheresis in order to obtain 200-300 mLplasma fraction enriched with peripheral blood mononuclear cells (PBMCs)for purification of leukemic cells (target yield 2.4×10⁹ cells) andmonocytes (target yield 5-30×10⁹ cells).

Transfection of autologous leukemic cells loaded with STAVs. Leukemiccells can be separately transfected (loaded) ex vivo with STAV1, STAV2,STAV3, STAV4 and STAV5, followed by UV irradiation and infused back intosubjects on Days 3, 17, 31, 45, and 59, respectively.

In situ DC maturation and re-infusion. DCs will be generated frommonocytes cultured for up to 7 days in the presence of GM-CSF and IL-4,and then loaded with mixture of dead STAVs loaded UV-irradiated leukemiccells on Day 8. In the next step, the immature DCs will be cultured for48 hours in the presence of maturation agents cocktail consisting ofTNF-α, and IL-1s. Then, matured DCs will be injected into subjects onDays 10, 17, 24, and 31.

Response assessment (Day 29+ or −7 days): Subjects with ATLL will beassessed by standard flow cytometry and TCR gene rearrangement studiesof peripheral blood, imaging studies (CT scan or CT-PET) (and bonemarrow biopsy to confirm complete response only). Subjects with AML willbe assessed by standard flow cytometry of peripheral blood (and bonemarrow biopsy with cytogenetic studies performed only to confirmcomplete response [(CR) or CR with incomplete hematologic recovery(CRi)]. Subjects with ALL will be assessed for minimal residual diseaseby standard flow cytometry of peripheral blood (and bone marrow biopsyto confirm complete response only). Standard PCR for bcr/abl may be usedin patients in Philadelphia chromosome positive (Ph+) patients toevaluate molecular response. All patients will be followed monthly forroutine monitoring and laboratory tests, and re-assessed for response atthe end of months 3, 6, 9 and 12 (+ or −7 days).

Response assessment (1 year): Subjects with a CR (or CRi) after Year 1who decide to remain on the study will be followed every 3 months (+ or−1 month) for 1 year for routine monitoring and laboratory tests, andresponse assessments. Subjects who progress after Year 1 will befollowed for survival only every 6 months (+ or − 1 month) via atelephone call from years 2 to 5.

Generation of DCs: DCs can be generated from monocytes cultured for upto 7 days in the presence of GM-CSF and IL-4, see also FIGS. 15B-15D.

FIG. 15A is a flow diagram showing a treatment protocol for treating apatient with cancer where a plurality of 200-300 mL plasma fractionenriched with PBMCs are obtained at day 1 902 from the patient's tumorand can be stored at −20° C., where one of the fractions is thawed andtransfected with a STAV (e.g., STAV1) 921, where the transfected cellsare irradiated with UV light (approximately 250 nm UV light for betweena lower limit of approximately 100 mJ/cm of UV irradiation and an upperlimit of approximately 200 mJ/cm of UV irradiation for between a lowerlimit of approximately 0.1 minute and an upper limit of approximately 10minutes) or otherwise prevented from proliferating (e.g., x-ray exposure0.75 Gy/min dose rate, 10-100 min, 50 keV effective energy) 931 andincubated for a period of time (e.g., 24 h) on day 2 941 and injectedinto the tumor on day 3 951. The procedure is repeated on day 15 withthe transfection of a second different STAV, (e.g., STAV2) 920 where thetransfected cells are irradiated with UV light 930 and incubated for aperiod of time (e.g., 24 h) on day 16 940 and injected into the tumor onday 17 950. The procedure can be repeated on day 29 with thetransfection of a third different STAV, (e.g., STAV3) 920 where thetransfected cells are irradiated with UV light 930 and incubated for aperiod of time (e.g., 24 h) on day 30 940 and injected into the tumor onday 31 950. The procedure can be repeated on day 43 with thetransfection of a fourth different STAV, (e.g., STAV4) 920 where thetransfected cells are irradiated with UV light 930 and incubated for aperiod of time (e.g., 24 h) on day 44 940 and injected into the tumor onday 45 950. The procedure can be repeated on day 57 with thetransfection of a fifth different STAV, (e.g., STAV5) 920 where thetransfected cells are irradiated with UV light 930 and incubated for aperiod of time (e.g., 24 h) on day 58 940 and injected into the tumor onday 59 950. The response to the treatment can be assessed on day 31, 91,181, 271 and 361 990, according to an embodiment of the invention. In anembodiment of the invention, if the response is sufficient the length oftime before the next administration of a dead leukemic fractiontransfected with a STAV can be extended or delayed.

FIG. 15B is a flow diagram showing a treatment protocol for treating apatient with cancer where a plurality of 200-300 mL plasma fractionenriched with PBMCs are obtained at day 1 from the patient's tumor andcan be stored at −20° C. On day 1 through to 7, monocytes are incubatedwith GM-CSF and IL4 911. On day 1 one of the leukemic cell fractions isthawed and transfected with a STAV (e.g., STAV1) 921, where thetransfected cells are irradiated with UV light (or otherwise preventedfrom proliferating) 931 and incubated for a period of time (e.g., 24 h)on day 2 941 and used to stimulate the immature DCs on day 8 961. On day8 the stimulated DCs loaded with the STAV are incubated with maturationagents 971. On day 10 the stimulated DCs loaded with the STAV and thematuration agents is injected into the tumor 981. On day 10 stimulatedimmature DCs loaded with a STAV are frozen (e.g., STAV2-STAV7) 962. Onday 17, 24, 31 the stimulated DCs loaded with a STAV (e.g., STAV2-STAV7)and the maturation agent are thawed and injected into the tumor 982. Theresponse to the treatment can be assessed on day 31, 91, 181, 271 and361 990, according to an embodiment of the invention. In an embodimentof the invention, if the response is sufficient the length of timebefore the next administration of DCs loaded with a STAV can be extendedor delayed.

FIG. 15C is a flow diagram showing a treatment protocol including theprotocol of treatment shown in FIG. 15A and the protocol of treatmentshown in FIG. 8B. On days 1-3, leukemic cells loaded with a STAV are UVirradiated and incubated for a period of time (e.g., 24 h) 940 andeither injected into the tumor on day 3 951 or used to generate thestimulated DC incubated with the maturation cocktail 970. On day 10 thestimulated DC loaded with a STAV and incubated with maturation agentsare injected into the tumor 981. On day 17, leukemic cells loaded with aSTAV are either injected into the tumor on 952 or used to generate thestimulated DC incubated with the maturation cocktail and are injectedinto the tumor 982. On day 24 the stimulated DC loaded with a STAV andincubated with maturation agents are injected into the tumor 983. On day31, leukemic cells loaded with a STAV are injected into the tumor on953. The response to the treatment can be assessed on day 31, 91, 181,271 and 361 990, according to an embodiment of the invention. In anembodiment of the invention, if the response is sufficient the length oftime before the next administration of a dead leukemic fractiontransfected with a STAV can be extended or delayed.

FIG. 15D is a flow diagram showing an alternative treatment protocol fora cancer requiring treatment with a plurality of doses of leukemic cellstreated with up to five STAVs comprising the group consisting of STAV1,STAV2, STAV3, STAV4 and STAV5 and a treatment with a plurality ofDendritic Cell vaccines generated with a plurality of STAVs selectedfrom the group consisting of STAV1, STAV2, STAV3, STAV4 and STAV5,according to an embodiment of the invention. On day 1, the leukemiccells are collected 901. On days 10, 17, 24, and 31 injection of thawedstimulated DCs is carried out 980. On days 3, 17, 31, 45, and 59injection of UV irradiated leukemic cells loaded with STAV1, STAV2,STAV3, STAV4 and STAV5 successively is carried out 980. Obtain 200-300mL plasma fraction enriched with PBMCs for purification of CD14+monocytes (target yield 5-30 ˜10⁹ cells) and separation of up to 2.4×10⁹leukemic cells 902. Fresh leukemic cells (˜3.6×10⁸ will be transfected(loaded) in vitro with STAV1, followed by UV irradiation on Day 3 920.Day 3: i.v. infusion of fresh UV-irradiated (dead) autologous leukemiccells transfected with STAV1 951. On the day of infusion, vital signswill be obtained prior and post infusion for toxicity monitoringpurposes. Remaining leukemic cells will be DMSO-frozen in 5 fractions(˜6×10⁸ cells for DC maturation and 3.6×10⁸ cells/vial for subsequenttransfection with STAV2, STAV3, STAV4, and STAV5, see 950. At the sametime, in vitro culture of autologous monocytes with GM-CSF+IL-4×7 dayscan be carried out to generate immature DCs 911. Days 8-10: in situ DCmaturation: the previously cultured immature DCs (6.0×10⁷ cells) will bestimulated with leukemic cells (6.0×10⁸) loaded with a mixture of STAV1,STAV2, STAV3, STAV4, and STAV5) in the presence of maturation agentscocktail consisting of TNF-α, IL-1s for 48 hours in order to generatemature DCs 970. Days 10, 17, 24, and 31 (+1 day): i.v. injections ofstimulated mature DCs (i.e., re-infusion of thawed mature DCs stimulatedwith UV-irradiated (dead) leukemic cells transfected with mixture ofSTAV1, STAV2, STAV3, STAV4, and STAV5) 980. On the day of infusion,vital signs will be obtained prior and post infusion for toxicitymonitoring purposes. Days 17, 31, 45, and 59 (Day 1+3 days): injectionsof UV-irradiated (dead) autologous leukemic cells (the yield of 3.6×10⁸cells after 48-hour culture) transfected two days prior with STAV2,STAV3, STAV4, and STAV5, respectively 950. While on-study subjects willreceive up to 5 separate doses of STAVs loaded cells (Days 3, 17, 31,45, and 59), and up to four (4) DC vaccinations (Days 10, 17, 24, and31). After therapy completion (approximately 2 months) subjects will befollowed at the end months 3, 6, 9, and 12 (+7 days) for clinicalassessment (complete physical exam, CBC, CMP, uric acid, phosphorus, andLDH), with response assessments as per Section 9.0 (CT scans, bonemarrow biopsy, peripheral blood flow cytometry, and PCR to evaluate forminimal residual disease). Those who remain progression-free after Year1 and decide to remain on study will be followed approximately every 3-6months during Year-2 post-treatment for routine monitoring andlaboratory tests, and response assessments at the discretion of theinvestigator. Thereafter, subjects will continue to be followed every 6months (+1 month) via a telephone call during Years 3 to 5 (at aminimum) for survival only with periodic visits and clinical assessmentsat the discretion of the investigator. Subjects who discontinuetreatment for disease progression will come off treatment and will befollowed for survival only every 6 months (+1 month) for up to 5 yearsfrom time of treatment initiation. Subjects who withdraw consent willcome off study. A study participant is considered to have completed thestudy once he or she completes all phases of the study treatment andstudy related laboratory tests. The primary and secondary endpoints willbe available for analysis once all patients have met the end points.Therefore, the clinical trial will be considered completed when the lastparticipant has completed all phases of the study including the lastvisit or the last scheduled procedure shown in the SoA, and the clinicalendpoints are available for analysis.

The proposed alternative treatment consists of combination of deadUV-irradiated STAVs loaded with autologous leukemic cells plus DCvaccine. Patients will receive up to 5 separate doses of STAVs loadedcells (Days 3, 17, 31, 45, and 59) 950, and up to four (4) DCvaccinations (Days 10, 17, 24, and 31) 980. The total treatment periodis two (2) months. The response is assessed on days 31, 91, 181, 271 and361 990.

FIG. 16 is a flow diagram showing a limiting toxicity protocol forrelapsed/refractory aggressive leukemia. In an embodiment of the presentinvention, enrollment of subjects of each cohort (ATLL, AML, ALL):enroll after the prior subject receives n doses of STAV1-STAVn loadedautologous leukemic cells and the (n−1) doses of DC vaccine withouttreatment limiting toxicities (TLTs) 1010. An Interim Safety Analysis isundertaken. 1020. If there is one patient with TLT 1030, then there isone patient with TLT 1030, continue staggered accrual until 3 straightsubjects have no treatment-limiting toxicity (TLT) 1050. If two or moresubjects have TLT, stop accrual and re-evaluate protocol to adjust fortoxicities and fix any other issues 1060. If there are no patients withTLT 1040, then continue injections of dead autologous STAVn loaded cellsand/or DC vaccinations in subjects 1070.

Days 3, 17, 31, 45, and 59: Sequential, i.v. infusion of fresh or thawedUV irradiated (dead) syngeneic leukemic cells transfected with fiveSTAVs selected from the group consisting of STAV1, STAV2, STAV3, STAV4,STAV5, STAV6 and STAV7.

Days 7-10: in situ DC maturation. Previously cultured immature DCs canbe stimulated (loaded) with mixture of thawed STAVs loaded leukemiccells for 24 hours in the presence of maturation agents cocktailconsisting of TNF-α and IL-1s for 48-72 hours in order to generatemature DCs days 10, 17, 24, and 31.

Re-infusion of mixture of thawed mature DCs stimulated with leukemiccells previously transfected with STAV2, STAV3, STAV4, and STAV5respectively.

Correlative Studies—Molecular evaluations/analysis in patients withHTLV-1/ATLL: Venous blood can be collected from patients diagnosed withleukemia-type HTLV-1/ATLL at baseline, Day 10, at the ends of Months 1,Month 3, Month, 6, Month, 9, Month 12, an at the end-of-treatment visitafter early discontinuation. Collected blood specimens can be processedand PMBCs can be isolated by centrifugation using standard Lymphoprep(ficol) procedure. A portion of fresh or thawed cells can be subjectedto magnetic CD4-enrichment by negative selection using commerciallyavailable kits. These cells can serve as source for protein and RNAafter standard extraction procedures. Non-enriched PBMCs can be used toextract genomic DNA for HTLV-1 pro-viral loads. The extracted cells maybe utilized fresh or be cryopreserved in DMSO-liquid nitrogen.

Re-infusion of dead STAVs-loaded HTLV-1/ATLL cells can lead tophagocytosis by APCs in vivo. Such event can result in excessindigestible STAVs that can activate STING dependent signaling withinAPCs which in turn can facilitate a potent anti-tumor T cell activation.In addition, APCs can present HTLV-1 antigens, such as HBZ (which isalways expressed ATLL tumors), which can in turn facilitate CTL primingagainst HTLV-1 infected cells and eliminate such clones.

CTL assays: To evaluate CTL responses after sequential administrationsof STAVs loaded tumor cells and DC vaccinations, venous blood can becollected from patients at baseline, before each DC vaccination on Days10, 17, 24, 31, 45, and at the end of Months 2, 3, and 6. Collectedblood specimens can be processed on the same day. PMBCs can be isolatedby centrifugation using standard Lymphoprep (ficol) procedure. Theextracted cells may be utilized fresh or be cryopreserved in DMSO-liquidnitrogen.

Methods: HTLV-1 specific CTL responses can be assessed using PBMCisolated from peripheral blood. CD8 T cells can be isolated using humanMACS CD8+ T cell isolation kit through negative selection (MiltenyilBiotec, 130-096-495). CD8 T Cells can be plated at 2×105 per well andstimulated with 20 μg/ml of tumor cell lysate protein or overlapping15-aa peptides covering the envelope, TAX or HBZ region of HTLV-1 forATLL (custom synthesized by GenScript). After 72 hours stimulation IFNgamma secreting cells can be determined using an ELISPOT assay for humanIFNγ and quantitated using a ELISPOT reader system. For flow cytometry,cells can be stimulated for 72 hours. Brefeldin A (3 mg/ml) can be addedto the cells 6h before analysis. Cells can be then washed, stained withcell surface marker (anti-CD3, anti-CD8), permeabilized withCytofix/Cytoperm (BD Biosciences), and stained with IFNγ. Data can beacquired using an LSR II flow cytometer.

B16 Melanoma Model

Sex matched C57/BL6 mice (n=10) inoculated with B16-OVA (5×10⁵) on theflanks. After 7, 10, and 13 days, when tumors are 50 mm³ in volume, 25μl (4 μg/mL; 0.1 μg/mouse) of Nano-STAVs (STAV1=(SEQ ID NO:24)+(SEQ IDNO:25); STAV2=(SEQ ID NO:26)+(SEQ ID NO:27); or STAV3=(SEQ IDNO:37)+(SEQ ID NO:38)] were injected i.t. in presence or absence ofanti-PD-1 or anti-PD-L1 (50 μg/mouse). Nanoparticles alone, checkpointinhibitor alone, PBS, isotype control antibody were used as controls.The tumor volume was measured using calipers and calculated with theformula V=(length×width²). The generation of anti-tumor CTL activity(against OVA) was measured.

The purity of all compounds was over 95% and was analyzed with WatersLC/MS system. ¹H NMR was obtained at 400 MHz. Chemical shifts arereported relative to dimethyl sulfoxide (6=2.50) for ¹H NMR. Data arereported as (br=broad, s=singlet, d=doublet, t=triplet, q=quartet,m=multiplet).

Abbreviations

AcOH: acetic acid; AEs: Adverse Events; ALL: Adult Lymphocytic Leukemia;AML: Acute Myeloid Leukemia; ANA: anti nuclear antibody; APC: AntigenPresenting Cells; ATLL: Adult T cell Lymphocytic Leukemia; ATM:atmosphere; BMDM: bone marrow derived macrophages; BP: Blood pressure;BOC₂O: di-tert-butyl dicarbonate; cGAMP: cyclic[G(2′,5′)pA(3′,5′)p];cGAS: cyclic guanosine monophosphate-adenosine monophosphate synthase;CNS: Central Nervous system; CR: Complete response; CTCAE: CommonTerminology Criteria for Adverse Events; CuSO₄: copper sulfate; CDCl₃:deuterated chloroform; CDN: cyclic dinucleotides; CTL: Cytotoxic Tcells; DC: dendritic cells; DCM: dichloromethane; DIEA:N,N-diisopropylethylamine; DMA: N,N-dimethylacetamide; DMAP:4-dimethylaminopyridine; DMF: N,N-dimethylformamide; DMSO: dimethylsulfoxide; DMSO-d₆: deuterated dimethyl sulfoxide; DOTAP:1,2-dioleoyl-3-trimethylammonium-propane; dsDNA: double strandeddeoxyribonucleic acid; DSPC: distearoylphosphatidylcholine; EoT: End ofTreatment; EDCI: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; ER:Endoplasmic Reticulum; ESI: electrospray ionization; EtOAc: ethylacetate; FAM: fluorescein; GMP: good manufacturing practice; H&E:hematoxylin and eosin; HR: Heart rate; HCl: hydrochloric acid; h:hour(s); HPLC: high-performance liquid chromatography; hTERT:immortalized human fibroblasts; HTLV-1: Human T-cell leukemia/lymphomavirus type 1; IFN: interferon; IRF3: Interferon Regulatory Factor 3;IRF7: Interferon Regulatory Factor 7; ISD: Interferon Stimulatory DNA;i.t.: intratumorally; i.v.: intravenous; J: Joule; LCMS: liquidchromatography-mass spectrometry; MS: median survival; MDA5: MelanomaDifferentiation associated Antigen 5; mL: milliliter; MeCN:acetonitrile; MHC: Major histocompatibility complex; MEF: MurineEmbryonic Fibroblasts; MeOH: methanol; mg: milligram; mmol: millimole;MgSO₄: magnesium sulfate; MHz: megahertz; min: minutes; MS: massspectrometry; MEF: Murine Embryonic Fibroblasts; Na₂CO₃: sodiumcarbonate; NaHCO₃: sodium bicarbonate; Nano-STAV a STAV in combinationwith a lipid nanoparticle; NF-kB: nuclear factorkappa-light-chain-enhancer of activated B cells; NMR: nuclear magneticresonance; PBMCs: Peripheral blood mononuclear cells; PCR: polymerasechain reaction; s.c.: subcutaneously; SEAP: secreted alkalinephosphatase; STAV: STING-dependent adjuvants; STING: Stimulator ofInterferon Genes; Tf: triflate; TKO: TREX1 KnockOut; TM: transmembrane;Pd₂(dba)₃ tris(dibenzylideneacetone)dipalladium(0); Pd(PPh₃)₂Cl₂:bis(triphenylphosphine)palladium(II) dichloride; PAMP: pathogenassociated molecular patterns; PE: Physical Exam; ppm: parts permillion; polyIC: Polyinosinic:polycytidylic acid; PCR: polymerase chainreaction; PTSA: para-toluene sulfonic acid; qPCR: quantitative real timePCR; QA: Quality Assurance; QC: Quality control; RIG-1: Retinoic acidInducible Gene 1; RNA: Ribonucleic Acid; RR: Respiratory rate; RT: roomtemperature; SOPs: Standard operating procedures; SPD: sum of theproduct of the diameters; STAVS: STING dependent adjuvants, activators;t-BuOH: tert-butanol; TBAF: tetra-n-butylammonium fluoride; TBK1:TANK-binding kinase 1; TCR: T cell receptor; THF: tetrahydrofuran; TRAP:translocon-associated protein; TFA: trifluoroacetic acid; TLR: Toll-likereceptors; TLT: Treatment Limiting Toxicity; TMS: trimethylsilane; TLC:thin layer chromatography; TSA: thermal shift assay; μL: microliter; UV:ultraviolet; VSV: vesicular stomatitis virus; WT: Wild Type.

Example 1

FIGS. 7A and 7B show flow diagrams of the protocol for administration ofNano-STAVs according to an embodiment of the present invention. Toexamine the importance of dose of STAV C57/BL6 mice (n=10) mice wereinoculated on both flanks with B16-OVA (5×10⁵) 620. After seven (7)days, when tumors were 50 mm³ in volume, 25 μl (4 μg/mL; 0.1 ug/mouse)or 25 μL (20 μg/mL; 0.5 μg/mouse for STAV dose escalation examination)of Nano-STAVs (comprising STAV1) was injected (on only one flank) i.t.630. Three (3) days later 25 μl (4 μg/mL; 0.1 ug/mouse) or 25 μL (20μg/mL; 0.5 μg/mouse for STAV dose escalation examination) of Nano-STAVs(comprising STAV2) was injected (on the same flank) i.t. 640. Finally,three (3) days later 25 μl (4 μg/mL; 0.1 ug/mouse) or 25 μL (20 μg/mL;0.5 μg/mouse for STAV dose escalation examination) of Nano-STAVs(comprising STAV3) was injected (on the same flank) i.t. 660.Nanoparticles alone, were used as controls. Body weights were monitoredbefore and after treatment and the tumor volume measured using calipersand calculated with the formula V=(length×width²) 622, 632, 662. Thegeneration of anti-tumor CTL activity (against OVA) was measured usingthe B16 model 621, 631, 661. Both flanks were monitored. Nano-STAVsgenerate effective anti-tumor T-cell responses which attack thenon-injected tumor on the opposite flank. In addition to these studies,serum taken from the mice, before every inoculation, ascertained theantibody response to the nanoparticles themselves (to gauge the immuneresponse to the formulations) 621, 631, 661. 0.5 μg of the particles wasused in solid-phase ELISA assays and the serum from immunized animalsincubated at 1/100 for 2 hours in PBS/0.1% Tween. Anti-murine conjugateswere used to detect any-anti-Nano-particle antibody. No significanthumoral response was observed to the Nano-STAV formulations.

FIGS. 7C and 7D show flow diagrams of the protocol for administration ofNano-STAVs with check point inhibitors according to an alternativeembodiment of the present invention. It is known that checkpointinhibitors can facilitate anti-tumor T cell activity. Nano-STAVs enterthe tumor microenvironment (TME) and function by entering and/oradhering to tumor cells. Tumor cells containing Nano-STAVs are engulfedby phagocytes in the TME, to activate extrinsic STING signaling andfacilitate the cross-presentation of tumor antigen. Accordingly,stimulating STING signaling is a key mechanism of cytotoxic T cellgeneration. Anti-PD-L1 and anti-PD1 (IgG BE0091 or anti-PD-L1 BE0101 oranti PD-1 J43 BE0033-2; BioXcell; 50 μg/mouse) was used to evaluatewhether Nano-STAVs exert synergistic effects with the checkpointinhibitors in syngeneic B16 melanoma model. Sex matched C57/BL6 mice(n=10) were inoculated with B16-OVA (5×10⁵) on both flanks 620. After 7,10, and 13 days, when tumors are 50 mm³ in volume, 25 μl (4 μg/mL; 0.1μg/mouse) of Nano-STAVs (comprising three or more of STAV1, STAV2,STAV3, STAV4, STAV5, STAV6 and/or STAV7) will be injected i.t. inpresence of anti-PD-1 or anti-PD-L1 (50 μg/mouse) 635, 645, 665.Nanoparticles alone, checkpoint inhibitor alone, PBS, isotype controlantibody were used as controls. The tumor volume was measured usingcalipers and calculated with the formula V=(length×width²). Nano-STAVsexhibit potent anti-tumor activity, increasing CTL infiltration withinthe TME and augment the efficacy of the PD-1/PD-L1 blockade.

Example 2

FIG. 1A shows confocal analysis of B16 OVA cells (B16) transfected withno DNA, labeled with FAM (green), DAPI (blue) and anti-calreticulin(red). FIG. 1B shows confocal analysis of B16 OVA cells (B16)transfected with STAVs-FAM, labeled with FAM (green), DAPI (blue) andanti-calreticulin (red). FIG. 2A is a line drawing of FIG. 1A showingconfocal analysis of B16 OVA cells (B16) transfected with no DNA labeledwith DAPI 205 and anti-calreticulin 210. FIG. 2B is a line drawing ofFIG. 1B showing confocal analysis of B16 OVA cells (B16) transfectedwith STAVs-FAM, labeled with FAM 215, DAPI 205 and anti-calreticulin210. The STAVs synthetically generated with exonuclease resistantphosphorothioates at the ends (ps) and greater than 70 base pairs wereeffective at stimulating STING-based cytokine production, regardless ofnucleotide content. Accordingly, it is possible to use one STAVs forprimary inoculation (AT rich) and a second STAVs for boosting purposes(GC) rich. In an embodiment of the invention, if the STAVs elicitedautoimmunity to the STAVs then this can allow a STAV based treatment toavoid autoimmune targeting of the STAV itself. Seven (7) STAVs have beenproposed to be used to select five (5) STavS for the purpose of Primingwith four (4) boosters available if necessary. FIG. 3A shows flowcytometry analysis of B16 OVA cells (B16) transfected with no DNA. FIG.3B shows flow cytometry analysis of B16 OVA cells (B16) transfected withSTAVs-FAM. As shown in FIG. 1B and FIG. 3B the fluorescent STAVstransfected into B16 melanoma are readily phagocytosed by APC's.

Example 3

The STAVs can be assembled into lipo-nanoparticles (referred to asNano-STAVs). However, the STAVs are much more stable than mRNA insertedinto LNPs. FIG. 4A shows a Transmission Electron Microscopy image ofNano-Empty LNPs at high magnification. FIG. 4B shows a TransmissionElectron Microscopy image of Nano-STAV LNPs at high magnification. FIG.4C shows a Transmission Electron Microscopy image of Nano-Empty LNPs atlow magnification. FIG. 4D shows a Transmission Electron Microscopyimage of Nano-STAV LNPs at low magnification. A Nano-STAVs formulatedwith phospholipid Distearoylphosphatidylcholine, Cholesterol,4-(dimethylamino)butanoate, and DMG-PEG 2000 has a size of approximately88 nm.

FIG. 4E shows a plot of cytokine expression for CXCL10 measured withqPCR in WT bone marrow derived mouse macrophages (11=PBS, 32=STAV,33=STAV+lipofectamine, 34=Nano-Empty, and 35=Nano-STAV). FIG. 4F shows aplot of cytokine expression for CXCL10 measured with qPCR in SKO bonemarrow derived mouse macrophages (11=PBS, 32=STAV,33=STAV+lipofectamine, 34=Nano-Empty, and 35=Nano-STAV). FIG. 4G shows aplot of cytokine expression for CXCL10 measured with qPCR in MAVS KObone marrow derived mouse macrophages (11=PBS, 32=STAV,33=STAV+lipofectamine, 34=Nano-Empty, and 35=Nano-STAV). FIG. 4H shows aplot of cytokine expression for IFN3 measured with ELISA in WT bonemarrow derived mouse macrophages (11=PBS, 32=STAV,33=STAV+lipofectamine, 34=Nano-Empty, and 35=Nano-STAV). FIG. 4I shows aplot of cytokine expression for IFN3 measured with ELISA in SKO bonemarrow derived mouse macrophages (11=PBS, 32=STAV,33=STAV+lipofectamine, 34=Nano-Empty, and 35=Nano-STAV). FIG. 4J shows aplot of cytokine expression for IFN3 measured with ELISA in MAVS KO bonemarrow derived mouse macrophages (11=PBS, 32=STAV,33=STAV+lipofectamine, 34=Nano-Empty, and 35=Nano-STAV). In wild-typemouse macrophages, the effect of Nano-STAVs on the antigen-presentingcells (APCs) was characterized by analyzing cytokine expression such asCXCL10 expression by quantitative real time PCR (qPCR) and IFN3 byELISA. Nano-STAVs were able to strongly induce the expression of CXCL10(FIG. 4E) and the secretion of IFN3 (FIG. 4H). However, this responsewas abolished in STING KO (FIG. 4F and FIG. 4I) cells but not in MAVS KOcells (FIG. 4G and FIG. 4J).

FIG. 4K shows a plot of cytokine expression for CXCL10 measured withqPCR in DCs (11=PBS, 32=STAV, 33=STAV+lipofectamine, 34=Nano-Empty, and35=Nano-STAV). FIG. 4L shows a plot of IFN3 measured with ELISA in DCs(11=PBS, 32=STAV, 33=STAV+lipofectamine, 34=Nano-Empty, and35=Nano-STAV). In DCs, the Nano-STAVs were able to strongly induce theexpression of CXCL10 (FIG. 4K) and the secretion of IFN3 (FIG. 4K),indicating that Nano-STAVs potently activate APCs in vitro throughSTING-dependent, RLR-independent signaling.

In an embodiment of the present invention, the Nano-STAVS can beformulated with DSPC, Cholesterol, ionizable MC3, and PEG-conjugatedlipid at a size of approximately 88 nm (FIGS. 4B and 4D). The effect ofNano-STAVs on the antigen-presenting cells (APCs) was characterized byanalyzing CXCL10 cytokine expression using quantitative real time PCR(qPCR) and IFN3 secretion using an ELISA assay.

Example 4

FIG. 5A shows tumor volume of mice s.c. injected with B16 OVA cells(5×10⁵ cells/mouse) on the right flank. On day 7, 10, and 13 after tumorinoculation, the mice were i.t. injected with 11=PBS, 32=STAV,34=Nano-Empty, and 35=Nano-STAV (0.1 μg/mouse). The tumor volume wasmeasured and calculated with the formula V=(length×width²)/2. At 17days, the spleen was extracted to measure IFNγ release from CD8+ Tcells. FIG. 5B shows digital photographs of mice treated as in FIG. 5A.FIG. 5C shows IFNγ-ELISPOT of (OVA−) mice treated as in FIG. 5A. FIG. 5Dshows IFNγ-ELISPOT of (OVA+) mice (i.e., s.c. injected with B16 OVAcells (5×10⁵ cells/mouse) on the right flank) and treated as in FIG. 5A.FIG. 5E shows tumor volume of mice treated as in FIG. 5A or 36=HSV1-γ34.5. Nano-STAVs are highly immunogenic and stimulate APC togenerate anti-tumor T cells in vivo as shown by the inoculation ofimmunocompetent C57BL/6 mice with B16 melanoma expressing OVA (B16 OVA).When the tumors were palpable, the mice were injected i.t. with 11=PBS,32=naked STAV, 34=Nano-Empty, or 35=Nano-STAV. Tumor sizes weremonitored, and the analysis of anti-tumor T cells response was measuredby IFNγ-ELISPOT. As shown in FIG. 5A and FIG. 5B the Nano-STAVs greatlyreduced the tumor growth compared to PBS control, nanoparticles empty(Nano-Empty), or STAVs alone. Comparison of FIG. 5C and FIG. 5Dindicates anti-OVA T cell activity in tumors inoculated with Nano-STAVs.This data indicates that Nano-STAVs are potentially immunogenic comparedto naked STAVs or Nano-Empty. Using the B16-OVA model, we comparedNano-STAVs therapy to oncolytic viral T-VEC therapy (using HSV1-γ34.5 asa model) that has been approved for the treatment of advanced melanoma,see FIG. 5E. Both Nano-STAV and HSV1-γ34.5 exert a strong reduction ofthe tumor growth compared to PBS, nanoparticles empty or STAVs alone(FIG. 5E). However, the Nano-STAV strategy is markedly improved overT-VEC therapy since Nano-STAVs are non-replicative and non-coding.

In an embodiment of the present invention, the Nano-STAVs can be highlyimmunogenic. In an embodiment of the present invention, the Nano-STAVscan stimulate APC to generate anti-tumor T cells in vivo. Inoculatedimmunocompetent C57BL/6 mice with B16 melanoma expressing OVA (B16 OVA)were injected i.t. with PBS, naked STAVs, Nano-empty, or Nano-STAVs whenthe tumors were palpable. Tumor sizes were monitored, and the analysisof anti-tumor T cells response was measured by IFNγ-ELISPOT.Unexpectedly, the Nano-STAVs greatly reduced tumor growth compared toPBS control, nanoparticles empty (Nano-Empty), or STAVs alone (FIGS. 5A,5B). That an additive effect was observed, where the improvementcompared with Mock for the STAVs alone and improvement compared withMock for the Nano-Empty alone was unexpected. Accordingly, using theNano-STAVs resulted in a synergistic increase in activity (i.e.,decrease in tumor volume). This synergistic effect is also observed inthe induction of IFNγ, which is also markedly increased, see FIG. 5C.Accordingly, Nano-STAVs are more immunogenic compared to naked STAVs orNano-Empty alone.

Using the B16-OVA model, Nano-STAVs therapy was compared with oncolyticviral T-VEC therapy (using HSV1-γ34.5 as a model) that was approved bythe FDA in 2015 for the treatment of advanced melanoma. Both Nano-STAVand HSV1-γ34.5 exert a strong reduction of the tumor growth compared toPBS, nanoparticles empty or STAVs alone (FIG. 5D). The Nano-STAVstrategy is markedly improved over T-VEC therapy since Nano-STAVs areimmunologically inert, non-coding, non-replicative, and therefore safer.Further, Nano-STAVs require less demanding quality control/GMP, and aretherefore less expensive to produce.

It is generally thought that T-VEC works by replicating in tumor cellscausing the tumor cells to lyse and release tumor antigens At the timeof this filling, the working hypothesis is that T-VEC exert its effectsthrough its dsDNA genome stimulating STING signaling in APC's ratherthan through an oncolytic, replicative activity. This hypothesis isbased on the understanding that T-VEC adheres to tumor cells but doesnot replicate efficiently or lyse them. Following phagocytosis of thetumor cell, the genome of T-VEC (a dsDNA linear genome of approximately150 kbp) activates STING signaling in the APC, to facilitate tumor cellantigen cross-presentation and the priming of T-cells. STING-deficientmice do not generate anti-tumor T cells following T-VEC treatment,indicating the importance of STING in this process. Since dsDNA ofapproximately 70 bp is sufficient to activate STING, the Nano-particledelivery of STAVs may exert an effect similar to T-VEC, butsignificantly more potent and safe.

A Nano-STAV strategy is markedly improved over T-VEC therapy sincepatients become seropositive against T-VEC (and other viral oncolytics)making the sequential delivery of this therapy ineffective. In contrast,a Nano-STAV strategy can sequentially deliver different STAVs (i.e.,prime, boost, boost) since the lipo-material is immunologically inertand the use of the different STAV formulations in our prime-booststrategy avoids auto-immune responses.

Evidence for the efficacy of the Nano-STAVs is also elicited from FIG.5D which shows no statistical difference between HSV1-γ34.5 andNano-STAV up until 15 days post treatment and at end of study (17 days)Nano-STAVs were only slightly inferior to treatment with the HSV1-γ34.5virus. This suggests that induction of IFNγ is an important diagnosticand reaffirms the importance of the synergistic effect observed for theNano-STAVs induction of IFNγ shown in FIG. 5C.

Example 5

FIG. 6A shows tumor volume of mice treated as in FIG. 5A with 11=PBS,32=STAV, 37=anti-PD1 (50 μg/mice) administered i.p., 34=Nano-Empty,38=Nano-Empty+anti-PD1 (50 μg/mice) administered i.p., 35=Nano-STAV (0.1μg/mouse), and 39=Nano-STAV+anti-PD1 (50 μg/mice) administered i.p. FIG.6B shows digital photographs of mice treated as in FIG. 6A. FIG. 6Cshows IFNγ-ELISPOT of (OVA−) mice treated as in FIG. 6A. FIG. 6D showsIFNγ-ELISPOT of (OVA+) mice (s.c. injected with B16 OVA cells (5×10⁵cells/mouse) on the right flank) and treated as in FIG. 6A.

Unexpectedly, Nano-STAVs exert greater activity in the presence ofcheckpoint inhibitors. Palpable tumors were inoculated with Nano-STAV orNano-Empty as described previously (see FIG. 6 ). One set of micereceived checkpoint therapy alone (50 μg/mouse×2 intraperitonealinjection of anti-PD1; CD279). A second set of mice received checkpointtherapy in combination with Nano-STAVs. A third set of mice receivedcheckpoint therapy with the Nano-Empty (control). Tumor sizes weremonitored, and the analysis of anti-tumor T cells responses measured byIFNγ-ELISPOT (see FIG. 6A and FIG. 6B). The Nano-STAVs exhibited robustsynergistic anti-tumor activity when used in combination with checkpointinhibitors when compared to each therapy alone (see FIGS. 6A-6D).

In an embodiment of the present invention, the Nano-STAVS exert potentanti-tumor activity following IT inoculation. In an alternativeembodiment of the present invention, the potent anti-tumor activityfollowing IT inoculation is greatly augmented in the presence ofcheckpoint inhibitors. In an embodiment of the present invention, theapproach is cost effective. In an embodiment of the present invention,the approach can be administered sequentially using different STAVs toboost anti-tumor activity. In an alternative embodiment of the presentinvention, the approach is compatible with and can be used to assistcheckpoint inhibitor therapy.

Example 6

All statistical analysis was performed by Student's t test. The datawere considered to be significantly different when P<0.05.

Further Embodiments

Embodiments contemplated herein include Embodiments P1-P98 following.

Embodiment P1. A composition for treating a human subject suffering froma cancer comprising a Nano-STAV including a double-stranded DNAincluding a first strand and a second strand, where the first strandcomprises at least eighty percent complimentary nucleobases with respectto the second strand, and a lipid nanoparticle including apolymer-conjugated lipid, a sterol, a phospholipid and an ionizinglipid.

Embodiment P2. The composition of Embodiment P1, where the first strandfurther includes at least one (1) exonuclease resistant phosphorothioate(ps) backbone moiety at the 5′ end and at least one (1) ps backbonemoiety at the 3′ end.

Embodiment P3. The composition of Embodiment P1, where the first strandfurther includes at least three (3) exonuclease resistantphosphorothioate (ps) backbone moieties at the 5′ end and at least three(3) ps backbone moiety at the 3′ end.

Embodiment P4. The composition of Embodiment P1, where the Nano-STAV isselected from the group consisting of STAV1=(SEQ ID NO:24) and (SEQ IDNO:25); STAV2=(SEQ ID NO:26) and (SEQ ID NO:27); STAV3=(SEQ ID NO:37)and (SEQ ID NO:38); STAV4=(SEQ ID NO:39)+(SEQ ID NO:40); STAV5=(SEQ IDNO:41)+(SEQ ID NO:42); STAV6=(SEQ ID NO:43)+(SEQ ID NO:44), andSTAV7=(SEQ ID NO:45)+(SEQ ID NO:46).

Embodiment P5. The composition of Embodiment P1, where thepolymer-conjugated lipid is DMG-PEG 2000.

Embodiment P6. The composition of Embodiment P1, where the sterol ischolesterol.

Embodiment P7. The composition of Embodiment P1, where the phospholipidis distearoylphosphatidylcholine.

Embodiment P8. The composition of Embodiment P1, where the ionizinglipid is selected from the group consisting of7-[(2-Hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl2-octyldecanoate,N,N-dimethyl-2,3-bis[(9Z)-9-octadecen-1-yloxy]-1-propanamine,N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethanamine,(6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate, 4-(dimethylamino)-butanoic acid,(10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-ylester, Heptadecan-9-yl8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, and[(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate).

Embodiment P9. The composition of Embodiment P1, where thedouble-stranded DNA comprises between a lower limit of sixty (60)nucleobases, and an upper limit of one hundred and twenty (120)nucleobases.

Embodiment P10. A composition for treating a human subject sufferingfrom a cancer including a Nano-STAV including a double-stranded DNAincluding a first strand and a second strand, where the first strandcomprises at least eighty percent complimentary nucleobases with respectto the second strand, and a lipid nanoparticle including apolymer-conjugated lipid, a sterol, a phospholipid, and an ionizinglipid or a cationic lipid.

Embodiment P11. The composition of Embodiment P10, where the firststrand further comprises at least one (1) exonuclease resistantphosphorothioate (ps) backbone moiety at the 5′ end and at least one (1)ps backbone moiety at the 3′ end.

Embodiment P12. The composition of Embodiment P10, where the firststrand further comprises at least three (3) exonuclease resistantphosphorothioate (ps) backbone moieties at the 5′ end and at least three(3) ps backbone moiety at the 3′ end.

Embodiment P13. The composition of Embodiment P10, where the Nano-STAVis selected from the group consisting of STAV1=(SEQ ID NO:24) and (SEQID NO:25); STAV2=(SEQ ID NO:26) and (SEQ ID NO:27); STAV3=(SEQ ID NO:37)and (SEQ ID NO:38); STAV4=(SEQ ID NO:39)+(SEQ ID NO:40); STAV5=(SEQ IDNO:41)+(SEQ ID NO:42); STAV6=(SEQ ID NO:43)+(SEQ ID NO:44), andSTAV7=(SEQ ID NO:45)+(SEQ ID NO:46).

Embodiment P14. The composition of Embodiment P10, where thepolymer-conjugated lipid is a polyethylene glycol (PEG)-conjugatedlipid.

Embodiment P15. The composition of Embodiment P14, where thePEG-conjugated lipid is selected from the group consisting of DMG-PEG2000 and DSPE PEG 2000.

Embodiment P16. The composition of Embodiment P10, where the ionizinglipid is selected from the group consisting of7-[(2-Hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl2-octyldecanoate,N,N-dimethyl-2,3-bis[(9Z)-9-octadecen-1-yloxy]-1-propanamine,N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethanamine,(6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate, 4-(dimethylamino)-butanoic acid,(10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-ylester, Heptadecan-9-yl8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, and[(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate).

Embodiment P17. The composition of Embodiment P10, where the cationiclipid is selected from the group consisting of1,2-dioleoyl-3-trimethylammonium-propane, dimethyldioctadecylammoniumbromide, 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol,dimethyldioctadecylammonium,1,2-dimyristoyl-3-trimethylammonium-propane,1,2-stearoyl-3-trimethylammonium-propane andN-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium.

Embodiment P18. The composition of Embodiment P10, where thedouble-stranded DNA comprises between a lower limit of sixty (60)nucleobases, and an upper limit of one hundred and twenty (120)nucleobases.

Embodiment P19. The composition of Embodiment P10, where the firststrand comprises at least eighty (80) percent of adenine nucleobases andthe second strand comprises at least eighty (80) percent of thyminenucleobases.

Embodiment P20. The composition of Embodiment P10, where the firststrand comprises at least eighty (80) percent of cytosine nucleobasesand the second strand comprises at least eighty (80) percent of guaninenucleobases.

Embodiment P21. The composition of Embodiment P10, where thedouble-stranded DNA comprises STAV1=(SEQ ID NO:24)+(SEQ ID NO:25).

Embodiment P22. The composition of Embodiment P10, where thedouble-stranded DNA comprises STAV2=(SEQ ID NO:26)+(SEQ ID NO:27).

Embodiment P23. The composition of Embodiment P10, where thedouble-stranded DNA comprises STAV3=(SEQ ID NO:37)+(SEQ ID NO:38).

Embodiment P24. The composition of Embodiment P10, where thedouble-stranded DNA comprises STAV4=(SEQ ID NO:39)+(SEQ ID NO:40).

Embodiment P25. The composition of Embodiment P10, where thedouble-stranded DNA comprises STAV5=(SEQ ID NO:41)+(SEQ ID NO:42).

Embodiment P26. The composition of Embodiment P10, where thedouble-stranded DNA comprises STAV6=(SEQ ID NO:43)+(SEQ ID NO:44).

Embodiment P27. The composition of Embodiment P10, where thedouble-stranded DNA comprises STAV7=(SEQ ID NO:45)+(SEQ ID NO:46).

Embodiment P28. A composition for treating a human subject sufferingfrom a cancer including a first Nano-STAV, where the first Nano-STAVincludes a first STAV selected from the group consisting of STAV1=(SEQID NO:24) and (SEQ ID NO:25); STAV2=(SEQ ID NO:26) and (SEQ ID NO:27);STAV3=(SEQ ID NO:37) and (SEQ ID NO:38); STAV4=(SEQ ID NO:39)+(SEQ IDNO:40); STAV5=(SEQ ID NO:41)+(SEQ ID NO:42); STAV6=(SEQ ID NO:43)+(SEQID NO:44), and STAV7=(SEQ ID NO:45)+(SEQ ID NO:46), and a LNP includinga polymer-conjugated lipid, a sterol, a phospholipid, and an ionizinglipid or a cationic lipid;

a second Nano-STAV, where the second Nano-STAV includes a second STAVselected from the group consisting of STAV1=(SEQ ID NO:24) and (SEQ IDNO:25); STAV2=(SEQ ID NO:26) and (SEQ ID NO:27); STAV3=(SEQ ID NO:37)and (SEQ ID NO:38); STAV4=(SEQ ID NO:39)+(SEQ ID NO:40); STAV5=(SEQ IDNO:41)+(SEQ ID NO:42); STAV6=(SEQ ID NO:43)+(SEQ ID NO:44), andSTAV7=(SEQ ID NO:45)+(SEQ ID NO:46), where the second STAV is not thefirst STAV, and the LNP.

Embodiment P29. The composition of Embodiment P28, where thePEG-conjugated lipid is selected from the group consisting of DMG-PEG2000 and DSPE PEG 2000.

Embodiment P30. The composition of Embodiment P27, where the sterol isselected from the group consisting of cholesterol, cholesterol sulfate,desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol,dihydrolanosterol, zymosterol, lathosterol, 14-demethyl-lanosterol,8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, FF-MAS, diosgenin,dehydroepiandrosterone (DHEA) sulfate, DHEA, sitosterol, lanosterol-95,zymostenol, sitostanol, campestanol, campesterol, 7-dehydrodesmosterol,pregnenolone, dihydro T-MAS, delta 5-avenasterol, brassicasterol,dihydro FF-MAS, 24-methylene cholesterol,3β-hydroxy-7-oxo-5-cholestenoic acid, 7α-hydroxy-3-oxo-4-cholestenoicacid, 3β,7α-dihydroxy-5-cholestenoic acid,3β,7β-dihydroxy-5-cholestenoic acid, 3β-hydroxy-5-cholestenoic acid,3-oxo-4-cholestenoic acid, 3β,7α,24S-trihydroxy-5-cholestenoic acid,3β,24S-dihydroxy-5-cholestenoic acid, 3β,7α,25-trihydroxy-5-cholestenoicacid, and 3β,25-OH-7-oxo-5-cholestenoic acid.

Embodiment P31. The composition of Embodiment P28, where thephospholipid is selected from the group consisting ofdistearoylphosphatidylcholine, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidic acid,phosphatidylinositol, phosphatidylglycerol, cardiolipin, dipalmitoyl,dimyristoyl, DSPC, dioleoyl, and L-α-phosphatidylcholine.

Embodiment P32. The composition of Embodiment P28, where the ionizinglipid is selected from the group consisting of7-[(2-Hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl2-octyldecanoate,N,N-dimethyl-2,3-bis[(9Z)-9-octadecen-1-yloxy]-1-propanamine,N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethanamine,(6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate, 4-(dimethylamino)-butanoic acid,(10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-ylester, Heptadecan-9-yl8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, and[(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate).

Embodiment P33. The composition of Embodiment P28, where the compositioninduces Cxcl10 and/or type I IFN cytokine production.

Embodiment P34. The composition of Embodiment P28, further includingadministering one or more of (i) a third STAV and the LNP, (ii) a fourthSTAV and the LNP, and (iii) a fifth STAV and the LNP, where the thirdSTAV, the fifth STAV and the fifth STAV are selected from the groupconsisting of STAV1=(SEQ ID NO:24) and (SEQ ID NO:25); STAV2=(SEQ IDNO:26) and (SEQ ID NO:27); STAV3=(SEQ ID NO:37) and (SEQ ID NO:38);STAV4=(SEQ ID NO:39)+(SEQ ID NO:40); STAV5=(SEQ ID NO:41)+(SEQ IDNO:42); STAV6=(SEQ ID NO:43)+(SEQ ID NO:44), and STAV7=(SEQ IDNO:45)+(SEQ ID NO:46), where the third STAV is not the second STAV,where the third STAV is not the first STAV, where the fourth STAV is notthe first STAV, where the fourth STAV is not the second STAV, where thefourth STAV is not the third STAV, where the fifth STAV is not the firstSTAV, where the fifth STAV is not the second STAV, where the fifth STAVis not the third STAV, where the fifth STAV is not the fourth STAV.

Embodiment P35. A kit for treating a STING deficiency in a mammalincluding a Nano-STAV including a STAV selected from the groupconsisting of STAV1 (SEQ ID NO:24 and SEQ ID NO:25), STAV2 (SEQ ID NO:26and SEQ ID NO:27), STAV3 (SEQ ID NO:37 and SEQ ID NO:38), STAV4 (SEQ IDNO:39 and SEQ ID NO:40), STAV5 (SEQ ID NO:41 and SEQ ID NO:42), STAV6(SEQ ID NO:43 and SEQ ID NO:44)), and STAV7=(SEQ ID NO:45)+(SEQ IDNO:46), and a LNP, and instructions for administering the Nano-STAV inthe mammal.

Embodiment P36. The kit of Embodiment P35, where the LNP includes apolymer-conjugated lipid, a sterol, a phospholipid, and an ionizinglipid.

Embodiment P37. The kit of Embodiment P36, where the polymer-conjugatedlipid is a polyethylene glycol (PEG)-conjugated lipid.

Embodiment P38. The kit of Embodiment P37, where the PEG-conjugatedlipid is selected from the group consisting of DMG-PEG 2000 and DSPE PEG2000.

Embodiment P39. The kit of Embodiment P36, where the sterol is selectedfrom the group consisting of cholesterol, cholesterol sulfate,desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol,dihydrolanosterol, zymosterol, lathosterol, 14-demethyl-lanosterol,8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, FF-MAS, diosgenin,dehydroepiandrosterone (DHEA) sulfate, DHEA, sitosterol, lanosterol-95,zymostenol, sitostanol, campestanol, campesterol, 7-dehydrodesmosterol,pregnenolone, dihydro T-MAS, delta 5-avenasterol, brassicasterol,dihydro FF-MAS, 24-methylene cholesterol,3β-hydroxy-7-oxo-5-cholestenoic acid, 7α-hydroxy-3-oxo-4-cholestenoicacid, 3β,7α-dihydroxy-5-cholestenoic acid,3β,7β-dihydroxy-5-cholestenoic acid, 3β-hydroxy-5-cholestenoic acid,3-oxo-4-cholestenoic acid, 3β,7α,24S-trihydroxy-5-cholestenoic acid,3β,24S-dihydroxy-5-cholestenoic acid, 3β,7α,25-trihydroxy-5-cholestenoicacid, and 3β,25-OH-7-oxo-5-cholestenoic acid.

Embodiment P40. The kit of Embodiment P36, where the phospholipid isselected from the group consisting of distearoylphosphatidylcholine,phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidic acid, phosphatidylinositol, phosphatidylglycerol,cardiolipin, dipalmitoyl, dimyristoyl, DSPC, dioleoyl, andL-α-phosphatidylcholine.

Embodiment P41. The kit of Embodiment P36, where the ionizing lipid isselected from the group consisting of7-[(2-Hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl2-octyldecanoate,N,N-dimethyl-2,3-bis[(9Z)-9-octadecen-1-yloxy]-1-propanamine,N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethanamine,(6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate, 4-(dimethylamino)-butanoic acid,(10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-ylester, Heptadecan-9-yl8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, and[(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate).

Embodiment P42. A kit for treating a STING deficiency in a mammalincluding a first Nano-STAV and a second Nano-STAV selected from thegroup consisting of a first STAV selected from the group consisting ofSTAV1 (SEQ ID NO:24 and SEQ ID NO:25), STAV2 (SEQ ID NO:26 and SEQ IDNO:27), STAV3 (SEQ ID NO:37 and SEQ ID NO:38), STAV4 (SEQ ID NO:39 andSEQ ID NO:40), STAV5 (SEQ ID NO:41 and SEQ ID NO:42), STAV6 (SEQ IDNO:43 and SEQ ID NO:44), and STAV7=(SEQ ID NO:45)+(SEQ ID NO:46), and alipid nanoparticle (LNP), and instructions for administering the firstNano-STAV and the second Nano-STAV.

Embodiment P43. The kit of Embodiment P42, where the LNP includes apolymer-conjugated lipid, a sterol, a phospholipid, and an ionizinglipid or a cationic lipid.

Embodiment P44. The kit of Embodiment P43, where the polymer-conjugatedlipid is selected from the group consisting of DMG-PEG 2000 and DSPE PEG2000.

Embodiment P45. The kit of claim 43, where the sterol is selected fromthe group consisting of cholesterol, cholesterol sulfate, desmosterol,stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol,zymosterol, lathosterol, 14-demethyl-lanosterol,8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, FF-MAS, diosgenin,dehydroepiandrosterone (DHEA) sulfate, DHEA, sitosterol, lanosterol-95,zymostenol, sitostanol, campestanol, campesterol, 7-dehydrodesmosterol,pregnenolone, dihydro T-MAS, delta 5-avenasterol, brassicasterol,dihydro FF-MAS, 24-methylene cholesterol,3β-hydroxy-7-oxo-5-cholestenoic acid, 7α-hydroxy-3-oxo-4-cholestenoicacid, 3β,7α-dihydroxy-5-cholestenoic acid,3β,7β-dihydroxy-5-cholestenoic acid, 3β-hydroxy-5-cholestenoic acid,3-oxo-4-cholestenoic acid, 3β,7α,24S-trihydroxy-5-cholestenoic acid,3β,24S-dihydroxy-5-cholestenoic acid, 3β,7α,25-trihydroxy-5-cholestenoicacid, and 3β,25-OH-7-oxo-5-cholestenoic acid.

Embodiment P46. The kit of Embodiment P43, where the phospholipid isselected from the group consisting of phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidic acid,phosphatidylinositol, phosphatidylglycerol, cardiolipin, dipalmitoyl,dimyristoyl, DSPC, dioleoyl, and L-α-phosphatidylcholine.

Embodiment P47. The kit of Embodiment P43, where the ionizing lipid isselected from the group consisting of7-[(2-Hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl2-octyldecanoate,N,N-dimethyl-2,3-bis[(9Z)-9-octadecen-1-yloxy]-1-propanamine,N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethanamine,(6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate, 4-(dimethylamino)-butanoic acid,(10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-ylester, Heptadecan-9-yl8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, and[(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate).

Embodiment P48. The kit of Embodiment P43, where the cationic lipid isselected from the group consisting of1,2-dioleoyl-3-trimethylammonium-propane, dimethyldioctadecylammoniumbromide, 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol,dimethyldioctadecylammonium,1,2-dimyristoyl-3-trimethylammonium-propane,1,2-stearoyl-3-trimethylammonium-propane andN-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium.

Embodiment P49. The kit of Embodiment P43, where the instructionsfurther comprise directions on one or both preparing and administering adendritic cell vaccine.

Embodiment P50. A method for treating a mammal suffering from a cancerincluding administering a first Nano-STAV and a second Nano-STAV to themammal, where a time period between administering the first Nano-STAVand the second Nano-STAV is between a minimum of approximately one day,and a maximum of approximately one month.

Embodiment P51. The method of Embodiment P50, where the first Nano-STAVand the second Nano-STAV are selected from the group consisting of STAV1(SEQ ID NO:24 and SEQ ID NO:25), STAV2 (SEQ ID NO:26 and SEQ ID NO:27),STAV3 (SEQ ID NO:37 and SEQ ID NO:38), STAV4 (SEQ ID NO:39 and SEQ IDNO:40), STAV5 (SEQ ID NO:41 and SEQ ID NO:42), STAV6 (SEQ ID NO:43 andSEQ ID NO:44), and STAV7=(SEQ ID NO:45)+(SEQ ID NO:46).

Embodiment P52. The method of Embodiment P51, further includingadministering a dendritic cell vaccine to the mammal.

Embodiment P53. A composition for treating a human subject sufferingfrom a cancer including a Nano-STAV, where the Nano-STAV includes adouble-stranded DNA, where each strand of DNA comprises at least one (1)exonuclease resistant phosphorothioate backbone moiety at the 5′ end andat least one (1) exonuclease resistant phosphorothioate backbone moietyat the 3′ end, and a LNP including DMG-PEG 2000; cholesterol;distearoylphosphatidylcholine, and(6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate.

Embodiment P54. The composition of Embodiment P53, where the Nano-STAVcomprises a STAV selected from the group consisting of STAV1 (SEQ IDNO:24 and SEQ ID NO:25), STAV2 (SEQ ID NO:26 and SEQ ID NO:27), STAV3(SEQ ID NO:37 and SEQ ID NO:38), STAV4 (SEQ ID NO:39 and SEQ ID NO:40),STAV5 (SEQ ID NO:41 and SEQ ID NO:42), STAV6 (SEQ ID NO:43 and SEQ IDNO:44), and STAV7=(SEQ ID NO:45)+(SEQ ID NO:46).

Embodiment P55. A method for treating a human subject suffering from acancer including infusing a plurality of incubated tumor cells loadedwith a first STAV into the human subject, and infusing a plurality ofmature DCs into the human subject, thereby treating the human subjectsuffering from the cancer.

Embodiment P56. The method of Embodiment P55, where generating theplurality of incubated tumor cells further comprises generating aplurality of dead tumor cells, where the plurality of dead tumor cellsare generated from a plurality of tumor cells treated to prevent cellproliferation.

Embodiment P57. The method of Embodiment P56, where the plurality oftumor cells are treated to prevent cell proliferation by exposing to UVfor between lower limit of approximately 10 mJ of UV irradiation, andupper limit of approximately 1 J of UV irradiation.

Embodiment P58. The method of Embodiment P56, where the plurality oftumor cells are treated to prevent cell proliferation by exposing tobetween a lower limit of approximately 240 nm UV light, and an upperlimit of approximately 300 nm UV light, for between a lower limit ofapproximately 100 mJ/cm of UV irradiation, and an upper limit ofapproximately 200 mJ/cm of UV irradiation, for between a lower limit ofapproximately 10⁻¹ minute, and an upper limit of approximately 10′minutes.

Embodiment P59. The method of Embodiment P55, where generating theplurality of incubated tumor cells further comprises generating aplurality of dead tumor cells, where the plurality of dead tumor cellsare generated from a plurality of tumor cells treated by exposing tox-rays.

Embodiment P60. The method of Embodiment P57, where generating theplurality of incubated tumor cells further comprises transfecting theplurality of dead tumor cells with a first STAV to generate theplurality of incubated tumor cells.

Embodiment P61. The method of Embodiment P60, where the first STAV isselected from the group consisting of STAV1 (SEQ ID NO:24 and SEQ IDNO:25), STAV2 (SEQ ID NO:26 and SEQ ID NO:27), STAV3 (SEQ ID NO:37 andSEQ ID NO:38), STAV4 (SEQ ID NO:39 and SEQ ID NO:40), STAV5 (SEQ IDNO:41 and SEQ ID NO:42), STAV6 (SEQ ID NO:43 and SEQ ID NO:44), andSTAV7=(SEQ ID NO:45)+(SEQ ID NO:46).

Embodiment P62. The method of Embodiment P60, where the plurality oftumor cells are treated to prevent cell proliferation beforetransfection with the first STAV.

Embodiment P63. The method of Embodiment P60, where the plurality oftumor cells are treated to prevent cell proliferation after transfectionwith the first STAV.

Embodiment P64. The method of Embodiment P55, where generating theplurality of mature DCs further comprises generating a plurality ofactivated DCs, where a plurality of DCs are cultured in the presence ofone or more activators to generate the plurality of activated DCs.

Embodiment P65. The method of Embodiment P64, where the one or moreactivators are selected from the group consisting of GM-CSF, IL-4,TNF-α, and IL-1s..

Embodiment P66. The method of Embodiment P64, where the plurality of DCsare cultured for between a lower limit of approximately 10 hours, and anupper limit of approximately 10 days.

Embodiment P67. The method of Embodiment P64, where generating theplurality of mature DCs further comprises generating a plurality ofimmature DCs, where the plurality of activated DCs are incubated with aplurality of UV-irradiated leukemic cells loaded with a second STAV togenerate the plurality of immature DCs.

Embodiment P68. The method of Embodiment P67, where the plurality ofactivated DCs are cultured for between a lower limit of approximately 1hour, and an upper limit of approximately 2 days.

Embodiment P69. The method of Embodiment P67, where the second STAV isselected from the group consisting of STAV1 (SEQ ID NO:24 and SEQ IDNO:25), STAV2 (SEQ ID NO:26 and SEQ ID NO:27), STAV3 (SEQ ID NO:37 andSEQ ID NO:38), STAV4 (SEQ ID NO:39 and SEQ ID NO:40), STAV5 (SEQ IDNO:41 and SEQ ID NO:42), STAV6 (SEQ ID NO:43 and SEQ ID NO:44), andSTAV7=(SEQ ID NO:45)+(SEQ ID NO:46).

Embodiment P70. The method of Embodiment P69, where the first STAV isselected as the second STAV.

Embodiment P71. The method of Embodiment P67, where generating theplurality of mature DCs further comprises culturing the plurality ofimmature DCs in the presence of one or more activators to generate theplurality of mature DCs.

Embodiment P72. The method of Embodiment P71, where the plurality ofimmature DCs are cultured for between a lower limit of approximately 1hour, and an upper limit of approximately 7 days.

Embodiment P72. A method for treating a mammal suffering from cancerincluding administering a first Nano-STAV to the mammal, waiting a timeperiod, and administering a second Nano-STAV to the mammal.

Embodiment P73. The method of Embodiment P71, where the one or moreactivators are selected from the group consisting of GM-CSF, IL-4,TNF-α, and IL-1s.

Embodiment P74. The method of Embodiment P55, further including a timeperiod between step (a) and step (b) of between a minimum ofapproximately one day, and a maximum of approximately one month.

Embodiment P75. The method of Embodiment P67, The method of claim 67,where the plurality of UV-irradiated leukemic cells are treated toprevent cell proliferation by exposing to between a lower limit ofapproximately 240 nm UV light, and an upper limit of approximately 300nm UV light, for between a lower limit of approximately 100 mJ/cm of UVirradiation, and an upper limit of approximately 200 mJ/cm of UVirradiation, for between a lower limit of approximately 10-1 minute, andan upper limit of approximately 10¹ minutes.

Embodiment P76. The method of Embodiment P64, further including wherethe plurality of activated DCs are incubated with a plurality of x-raytreated leukemic cells to prevent cell proliferation loaded with asecond STAV to generate a plurality of immature DCs.

Embodiment P77. A method for treating a human subject suffering from acancer including infusing a plurality of first incubated tumor cellsloaded with a first STing dependent ActiVator (STAV) into the humansubject, infusing a plurality of mature dendritic cells (DCs) into thehuman subject, and infusing a plurality of second incubated tumor cellsloaded with a second STAV into the human subject, thereby treating thehuman subject suffering from the cancer.

Embodiment P78. The method of Embodiment P77, where the second STAV isnot the first STAV.

Embodiment P79. The method of Embodiment P77, where generating theplurality of first incubated tumor cells and/or the plurality of secondincubated tumor cells further comprises generating a plurality of deadtumor cells, where the plurality of dead tumor cells are generated froma plurality of tumor cells treated to prevent cell proliferation.

Embodiment P80. The method of Embodiment P79, where the plurality oftumor cells are treated to prevent cell proliferation by exposing tobetween a lower limit of approximately 240 nm UV light, and an upperlimit of approximately 300 nm UV light, for between a lower limit ofapproximately 100 mJ/cm of UV irradiation, and an upper limit ofapproximately 200 mJ/cm of UV irradiation, for between a lower limit ofapproximately 10-1 minute, and an upper limit of approximately 10¹minutes.

Embodiment P81. The method of Embodiment P79, where generating theplurality of first incubated tumor cells and the plurality of secondincubated tumor cells further comprises transfecting the plurality ofdead tumor cells with a first STAV and a second STAV to generate aplurality of first transfected tumor cells and a plurality of secondtransfected tumor cells.

Embodiment P82. The method of Embodiment P81, where the first STAV andthe second STAV are selected from the group consisting of STAV1 (SEQ IDNO:24 and SEQ ID NO:25), STAV2 (SEQ ID NO:26 and SEQ ID NO:27), STAV3(SEQ ID NO:37 and SEQ ID NO:38), STAV4 (SEQ ID NO:39 and SEQ ID NO:40),STAV5 (SEQ ID NO:41 and SEQ ID NO:42), STAV6 (SEQ ID NO:43 and SEQ IDNO:44), and STAV7=(SEQ ID NO:45)+(SEQ ID NO:46), where the first STAV isnot the second STAV.

Embodiment P83. The method of Embodiment P81, where generating theplurality of first incubated tumor cells and the plurality of secondincubated tumor cells further comprises incubating the plurality offirst transfected tumor cells and the plurality of second transfectedtumor cells to generate the plurality of first incubated tumor cells andthe plurality of second incubated tumor cells.

Embodiment P84. The method of Embodiment P77, where generating theplurality of mature DCs further comprises generating a plurality ofactivated DCs, where a plurality of DCs are cultured in the presence ofone or more activators to generate the plurality of activated DCs.

Embodiment P85. The method of Embodiment P64, where the one or moreactivators are selected from the group consisting of GM-CSF, IL-4,TNF-α, and IL-1s.

Embodiment P86. The method of Embodiment P84, where the plurality of DCsare cultured for between a lower limit of approximately 10 hours, and anupper limit of approximately 10 days.

Embodiment P87. The method of Embodiment P84, where generating theplurality of mature DCs further comprises generating a plurality ofimmature DCs, where the plurality of activated DCs are incubated with aplurality of UV-irradiated leukemic cells loaded with a second STAV togenerate the plurality of immature DCs.

Embodiment P88. The method of Embodiment P87, where the plurality ofactivated DCs are cultured for between a lower limit of approximately 1hour, and an upper limit of approximately 2 days.

Embodiment P89. The method of Embodiment P67, where the second STAV isselected from the group consisting of STAV1 (SEQ ID NO:24 and SEQ IDNO:25), STAV2 (SEQ ID NO:26 and SEQ ID NO:27), STAV3 (SEQ ID NO:37 andSEQ ID NO:38), STAV4 (SEQ ID NO:39 and SEQ ID NO:40), STAV5 (SEQ IDNO:41 and SEQ ID NO:42), STAV6 (SEQ ID NO:43 and SEQ ID NO:44), andSTAV7=(SEQ ID NO:45)+(SEQ ID NO:46).

Embodiment P90. The method of Embodiment P89, where the first STAV isselected as the second STAV.

Embodiment P91. The method of Embodiment P87, where generating theplurality of mature DCs further comprises culturing the plurality ofimmature DCs in the presence of one or more activators to generate theplurality of mature DCs.

Embodiment P92. The method of Embodiment P91, where the plurality ofimmature DCs are cultured for between a lower limit of approximately 1hour, and an upper limit of approximately 7 days.

Embodiment P93. The method of Embodiment P91, where the one or moreactivators are selected from the group consisting of GM-CSF, IL-4,TNF-α, and IL-1s.

Embodiment P94. The method of Embodiment P77, further including a firsttime period between step (a) and step (b) of between a minimum ofapproximately one day, and a maximum of approximately one month.

Embodiment P95. The method of Embodiment P94, The method of claim 94,further including a second time period between step (b) and step (c) ofbetween a minimum of approximately one day, and a maximum ofapproximately one month.

Embodiment P96. The method of Embodiment P87, The method of claim 87,where the plurality of UV-irradiated leukemic cells are treated toprevent cell proliferation by exposing to between a lower limit ofapproximately 240 nm UV light, and an upper limit of approximately 300nm UV light, for between a lower limit of approximately 100 mJ/cm of UVirradiation, and an upper limit of approximately 200 mJ/cm of UVirradiation, for between a lower limit of approximately 10⁻¹ minute, andan upper limit of approximately 10¹ minutes

Embodiment P97. A composition for treating a human subject sufferingfrom a cancer including a first STAV, and a second STAV, where the firstSTAV and the second STAV are selected from the group consisting of STAV1(SEQ ID NO:24 and SEQ ID NO:25), STAV2 (SEQ ID NO:26 and SEQ ID NO:27),STAV3 (SEQ ID NO:37 and SEQ ID NO:38), STAV4 (SEQ ID NO:39 and SEQ IDNO:40), STAV5 (SEQ ID NO:41 and SEQ ID NO:42), STAV6 (SEQ ID NO:43 andSEQ ID NO:44), and STAV7=(SEQ ID NO:45)+(SEQ ID NO:46).

Embodiment P98. The composition of Embodiment P97, further including adendritic cell vaccine.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the present application.

Example embodiments of the methods, systems, and components of thepresent invention have been described herein. As noted elsewhere, theseexample embodiments have been described for illustrative purposes only,and are not limiting. Other embodiments are possible and are covered bythe invention. Such embodiments will be apparent to persons skilled inthe relevant art(s) based on the teachings contained herein. Forexample, it is envisaged that, irrespective of the actual shape depictedin the various Figures and embodiments described above, the outerdiameter exit of the inlet tube can be tapered or non-tapered and theouter diameter entrance of the outlet tube can be tapered ornon-tapered.

Thus, the breadth and scope of the present invention should not belimited by any of the above-described exemplary embodiments, but shouldbe defined only in accordance with the following claims and theirequivalents.

What is claimed:
 1. A composition for treating a human subject sufferingfrom a cancer with a STing dependent ActiVator (STAV) lipid nanoparticle(LNP) comprising: a first Nano-STAV comprising: a) a double-stranded DNAcomprising: a first strand and a second strand, where the first strandcomprises at least eighty (80) percent complimentary nucleobases withrespect to the second strand; and b) a LNP comprising: apolymer-conjugated lipid; a sterol; a phospholipid; and an ionizinglipid or a cationic lipid.
 2. The composition of claim 1, where one orboth the first strand and the second strand further comprise at leastone (1) modification located at one or both a 5′ end and a 3′ end. 3.The composition of claim 2, where the modification comprises anexonuclease resistant phosphorothioate backbone moiety.
 4. Thecomposition of claim 1, where the first Nano-STAV is selected from thegroup consisting of the first strand is SEQ ID NO:24 and the secondstrand is SEQ ID NO:25, the first strand is SEQ ID NO:26 and the secondstrand is SEQ ID NO:27, the first strand is SEQ ID NO:37 and the secondstrand is SEQ ID NO:38, the first strand is SEQ ID NO:39 and the secondstrand is SEQ ID NO:40, the first strand is SEQ ID NO:41 and the secondstrand is SEQ ID NO:42, the first strand is SEQ ID NO:43 and the secondstrand is SEQ ID NO:44, and the first strand is SEQ ID NO:45 and thesecond strand is SEQ ID NO:46.
 5. A kit for treating a STING deficiencyin a mammal with a STing dependent ActiVator (STAV) lipid nanoparticle(LNP) comprising: (A) a first Nano-STAV comprising: (i) a firstdouble-stranded DNA comprising: a first strand and a second strand,where the first strand comprises at least eighty percent complimentarynucleobases with respect to the second strand; and (ii) a LNPcomprising: a polymer-conjugated lipid; a sterol; a phospholipid; and anionizing lipid or a cationic lipid; (B) a second Nano-STAV comprising:(iii) a second double-stranded DNA comprising: a third strand and afourth strand, where the third strand comprises at least eighty percentcomplimentary nucleobases with respect to the fourth strand, where thethird strand is not the first strand, where the fourth strand is not thesecond strand; and (iv) the LNP; and (C) instructions for administeringthe first Nano-STAV and the second Nano-STAV in the mammal.
 6. The kitof claim 5, where one or both the first strand and the second strandfurther comprise at least one (1) modification located at one or both a5′ end and a 3′ end.
 7. The kit of claim 6, where the modificationcomprises an exonuclease resistant phosphorothioate (ps) backbonemoiety.
 8. The kit of claim 5, where the first Nano-STAV is selectedfrom the group consisting of the first strand is SEQ ID NO:24 and thesecond strand is SEQ ID NO:25, the first strand is SEQ ID NO:26 and thesecond strand is SEQ ID NO:27, the first strand is SEQ ID NO:37 and thesecond strand is SEQ ID NO:38, the first strand is SEQ ID NO:39 and thesecond strand is SEQ ID NO:40, the first strand is SEQ ID NO:41 and thesecond strand is SEQ ID NO:42, the first strand is SEQ ID NO:43 and thesecond strand is SEQ ID NO:44, and the first strand is SEQ ID NO:45 andthe second strand is SEQ ID NO:46.
 9. The kit of claim 5, where thesecond Nano-STAV is selected from the group consisting of the thirdstrand is SEQ ID NO:24 and the fourth strand is SEQ ID NO:25, the thirdstrand is SEQ ID NO:26 and the fourth strand is SEQ ID NO:27, the thirdstrand is SEQ ID NO:37 and the fourth strand is SEQ ID NO:38, the thirdstrand is SEQ ID NO:39 and the fourth strand is SEQ ID NO:40, the thirdstrand is SEQ ID NO:41 and the fourth strand is SEQ ID NO:42, the thirdstrand is SEQ ID NO:43 and the fourth strand is SEQ ID NO:44, and thethird strand is SEQ ID NO:45 and the fourth strand is SEQ ID NO:46. 10.A method for treating a mammal suffering from a cancer comprising (A)administering at a first time a first Nano-STAV comprising: (i) a firstdouble-stranded DNA comprising: a first strand and a second strand,where the first strand comprises at least eighty percent complimentarynucleobases with respect to the second strand; and (ii) a LNPcomprising: a polymer-conjugated lipid; a sterol; a phospholipid; and anionizing lipid or a cationic lipid; (B) administering at a second time asecond Nano-STAV, where the second time is between: a minimum ofapproximately one day; and a maximum of approximately one month afterthe first time, the second Nano-STAV comprising: (iii) a seconddouble-stranded DNA comprising: a third strand and a fourth strand,where the third strand comprises at least eighty percent complimentarynucleobases with respect to the fourth strand, where the third strand isnot the first strand, where the fourth strand is not the second strand;and (iv) the LNP.
 11. The method of claim 10, where one or both thefirst strand and the second strand further comprise at least one (1)modification located at one or both a 5′ end and a 3′ end.
 12. Themethod of claim 11, where the modification comprises an exonucleaseresistant phosphorothioate backbone moiety.
 13. The method of claim 10,where the first Nano-STAV is selected from the group consisting of thefirst strand is SEQ ID NO:24 and the second strand is SEQ ID NO:25, thefirst strand is SEQ ID NO:26 and the second strand is SEQ ID NO:27, thefirst strand is SEQ ID NO:37 and the second strand is SEQ ID NO:38, thefirst strand is SEQ ID NO:39 and the second strand is SEQ ID NO:40, thefirst strand is SEQ ID NO:41 and the second strand is SEQ ID NO:42, thefirst strand is SEQ ID NO:43 and the second strand is SEQ ID NO:44, andthe first strand is SEQ ID NO:45 and the second strand is SEQ ID NO:46.14. The method of claim 10, where the second Nano-STAV is selected fromthe group consisting of the third strand is SEQ ID NO:24 and the fourthstrand is SEQ ID NO:25; the third strand is SEQ ID NO:26 and the fourthstrand is SEQ ID NO:27, the third strand is SEQ ID NO:37 and the fourthstrand is SEQ ID NO:38, the third strand is SEQ ID NO:39 and the fourthstrand is SEQ ID NO:40, the third strand is SEQ ID NO:41 and the fourthstrand is SEQ ID NO:42, the third strand is SEQ ID NO:43 and the fourthstrand is SEQ ID NO:44, and the third strand is SEQ ID NO:45 and thefourth strand is SEQ ID NO:46.
 15. The method of claim 10, furthercomprising administering one or both a first DC (Dendritic Cell) vaccineand a second DC vaccine to the mammal.
 16. The method of claim 15, wherethe first DC vaccine comprises UV-irradiated leukemic cells loaded withthe first nano-STAV and the second DC vaccine comprises UV-irradiatedleukemic cells loaded with the second nano-STAV.
 17. The method of claim16, where one or both the first DC vaccine and the second DC vaccinefurther comprise culturing UV-irradiated leukemic cells with one or moreactivators.
 18. The method of claim 17, where the one or more activatorsare selected from the group consisting of TNF-α, and IL-1β.
 19. Themethod of claim 16, where the first DC vaccine is administered after thefirst time and before the second time.
 20. The method of claim 16, wherethe second DC vaccine is administered after the second time.